Focused libraries of genetic packages

ABSTRACT

Focused libraries of vectors or genetic packages that display, display and express, or comprise a member of a diverse family of antibody peptides, polypeptides or proteins and collectively display, display and express, or comprise at least a portion of the focused diversity of the family. The libraries have length and sequence diversities that mimic that found in native human antibodies.

This application claims the benefit under 35 USC § 120 of U.S.provisional application 60/256,380, filed Dec. 18, 2001. The provisionalapplication and the Tables attached to it are specifically incorporatedby reference herein.

The present invention relates to focused libraries of genetic packagesthat each display, display and express, or comprise a member of adiverse family of peptides, polypeptides or proteins and collectivelydisplay, display and express, or comprise at least a portion of thefocused diversity of the family. The focused diversity of the librariesof this invention comprises both sequence diversity and lengthdiversity. In a preferred embodiment, the focused diversity of thelibraries of this invention is biased toward the natural diversity ofthe selected family. In a more preferred embodiment, the libraries arebiased toward the natural diversity of human antibodies and arecharacterized by variegation in their heavy chain and light chaincomplementarity determining regions (“CDRs”).

The present invention further relates to vectors and genetic packages(e.g., cells, spores or viruses) for displaying, or displaying andexpressing a focused diverse family of peptides, polypeptides orproteins. In a preferred embodiment the genetic packages are filamentousphage or phagemids or yeast. Again, the focused diversity of the familycomprises diversity in sequence and diversity in length.

The present invention further relates to methods of screening thefocused libraries of the invention and to the peptides, polypeptides andproteins identified by such screening.

BACKGROUND OF THE INVENTION

It is now common practice in the art to prepare libraries of geneticpackages that individually display, display and express, or comprise amember of a diverse family of peptides, polypeptides or proteins andcollectively display, display and express, or comprise at least aportion of the amino acid diversity of the family. In many commonlibraries, the peptides, polypeptides or proteins are related toantibodies (e.g., single chain Fv (scFv), Fv, Fab, whole antibodies orminibodies (i.e., dimers that consist of V_(H) linked to V_(L))). Often,they comprise one or more of the CDRs and framework regions of the heavyand light chains of human antibodies.

Peptide, polypeptide or protein libraries have been produced in severalways in the prior art. See e.g., Knappik et al., J. Mol. Biol., 296, pp.57-86 (2000), which is incorporated herein by references. One method isto capture the diversity of native donors, either naive or immunized.Another way is to generate libraries having synthetic diversity. A thirdmethod is a combination of the first two. Typically, the diversityproduced by these methods is limited to sequence diversity, i.e., eachmember of the library differs from the other members of the family byhaving different amino acids or variegation at a given position in thepeptide, polypeptide or protein chain. Naturally diverse peptides,polypeptides or proteins, however, are not limited to diversity only intheir amino acid sequences. For example, human antibodies are notlimited to sequence diversity in their amino acids, they are alsodiverse in the lengths of their amino acid chains.

For antibodies, diversity in length occurs, for example, during variableregion rearrangements. See e.g., Corbett et al., J. Mol. Biol., 270, pp.587-97 (1997). The joining of V genes to J genes, for example, resultsin the inclusion of a recognizable D segment in CDR3 in about half ofthe heavy chain antibody sequences, thus creating regions encodingvarying lengths of amino acids. The following also may occur duringjoining of antibody gene segments: (i) the end of the V gene may havezero to several bases deleted or changed; (ii) the end of the D segmentmay have zero to many bases removed or changed; (iii) a number of randombases may be inserted between V and D or between D and J; and (iv) the5′ end of J may be edited to remove or to change several bases. Theserearrangements result in antibodies that are diverse both in amino acidsequence and in length.

Libraries that contain only amino acid sequence diversity are, thus,disadvantaged in that they do not reflect the natural diversity of thepeptide, polypeptide or protein that the library is intended to mimic.Further, diversity in length may be important to the ultimatefunctioning of the protein, peptide or polypeptide. For example, withregard to a library comprising antibody regions, many of the peptides,polypeptides, proteins displayed, displayed and expressed, or comprisedby the genetic packages of the library may not fold properly or theirbinding to an antigen may be disadvantaged, if diversity both insequence and length are not represented in the library.

An additional disadvantage of prior art libraries of genetic packagesthat display, display and express, or comprise peptides, polypeptidesand proteins is that they are not focused oh those members that arebased on natural occurring diversity and thus on members that are mostlikely to be functional. Rather, the prior art libraries, typically,attempt to include as much diversity or variegation at every amino acidresidue as possible. This makes library construction time-consuming andless efficient than possible. The large number of members that areproduced by trying to capture complete diversity also makes screeningmore cumbersome than it needs to be. This is particularly true giventhat many members of the library will not be functional.

SUMMARY OF THE INVENTION

One objective of this invention is focused libraries of vectors orgenetic packages that encode members of a diverse family of peptides,polypeptides or proteins wherein the libraries encode populations thatare diverse in both length and sequence. The diverse length comprisingcomponents that contain motifs that are likely to fold and function inthe context of the parental peptide, polypeptide or protein.

Another object of this invention is focused libraries of geneticpackages that display, display and express, or comprise a member of adiverse family of peptides, polypeptides and proteins and collectivelydisplay, display and express, or comprise at least a portion of thefocused diversity of the family. These libraries are diverse not only intheir amino acid sequences, but also in their lengths. And, theirdiversity is focused so as to more closely mimic or take into accountthe naturally-occurring diversity of the specific family that thelibrary represents.

Another object of this invention is diverse, but focused, populations ofDNA sequences encoding peptides, polypeptides or proteins suitable fordisplay or display and expression using genetic packages (such as phageor phagemids) or other regimens that allow selection of specific bindingcomponents of a library.

A further object of this invention is focused libraries comprising theCDRs of human antibodies that are diverse in both their amino acidsequence and in their length (examples of such libraries includelibraries of single chain Fv (scFv), Fv, Fab, whole antibodies orminibodies (i.e., dimers that consist of V_(H) linked to V_(L))). Suchregions may be from the heavy or light chains or both and may includeone or more of the CDRs of those chains. More preferably, the diversityor variegation occurs in all of the heavy chain and light chain CDRs.

It is another object of this invention to provide methods of making andscreening the above libraries and the peptides, polypeptides andproteins obtained in such screening.

Among the preferred embodiments of this invention are the following:

1. A focused library of vectors or genetic packages that display,display and express, or comprise a member of a diverse family of humanantibody related peptides, polypeptides and proteins and collectivelydisplay, display and express, or comprise at least a portion of thediversity of the antibody family, the vectors or genetic packages beingcharacterized by variegated DNA sequences that encode a heavy chain CDR1selected from the group consisting of:

-   -   (1) <1>₁Y₂<1>₃M₄<1>₅, wherein <1> is an equimolar mixture of        each of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P,        Q, R, S, T, V, W, and Y;    -   (2) (S/T)₁(S/G/X)₂(S/G/X)₃Y₄Y₅W₆(S/G/X)₇, wherein (S/T) is a 1:1        mixture of S and T residues, (S/G/X) is a mixture of 0.2025 S,        0.2025 G and 0.035 of each of amino acid residues A, D, E, F, H,        I, K, L, M, N, P, Q, R, T, V, W, and Y;    -   (3) V₁S₂G₃G₄S₅I₆S₇<1>₈<1>₉<1>₁₀Y₁₁Y₁₂W₁₃<1>₁₄, wherein <1> is an        equimolar mixture of each of amino acid residues A, D, E, F, G,        H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and    -   (4) mixtures of vectors or genetic packages characterized by any        of the above DNA sequences, preferably in the ratio: HC CDR1s        (1):(2):(3)::0.80:0.17:0.02.

2. A focused library of vectors or genetic packages that display,display and express, or comprise a member of a diverse family of humanantibody related peptides, polypeptides and proteins and collectivelydisplay, display and express, or comprise at least a portion of thediversity of the antibody facility, the vectors or genetic packagesbeing characterized by variegated DNA sequences that encode a heavychain CDR2 selected from the group consisting of:

-   -   (1) <2>I<2><3>SGG<1>T<1>YADSVKG, wherein <1> is an equimolar        mixture of each of amino acid residues A, D, E, F, G, H, I, K,        L, M, N, P, Q, R, S, T, V, W, and Y; <2> is an equimolar mixture        of each of amino acid residues Y, R, W, V, G, and S; and <3> is        an equimolar mixture of each of amino acid residues P, S, and G        or an equimolar mixture of P and S;    -   (2) <1>I<4><1><1><G><5><1><1><1>YADSVKG, herein <1> is an        equimolar mixture of each of amino acid residues A, D, E, F, G,        H, I, K, L, M, N, P, Q, R, S, T, V, W, and Y; <4> is an        equimolar mixture of residues D, I, N, S, W, Y; and <5> is an        equimolar mixture of residues S, G, D and N;    -   (3) <1>I<4><1><1>G<5><1><1>YNPSLKG, wnerein <1> is an equimolar        mixture of each of amino acid residues A, D, E, F, G, H, I, K,        L, M, N, P, Q, R, S, T, V, W and Y; and <4> and <5> are as        defined above;    -   (4) <1>I<8>S<1><1><1>GGYY<1>YAASVKG, wherein <1> is an equimolar        mixture of each amino acid residues A, D, E, F, G, H, I, K, L,        M, N, P, Q, R, S, T, V, W and Y; <8> is 0.27 R and 0.027 of each        of ADEFGHIKLMNPQSTVWY; and    -   (5) mixtures of vectors or genetic packages characterized by any        of the above DNA sequences, preferably in the ratio: HC CDR2s:        (1)/(2) (equimolar): (3):(4)::0.54:0.43:0.03.

3. A focused library of vectors or genetic packages that display,display and express, or comprise a member of a diverse family of humanantibody related peptides, polypeptides and proteins and collectivelydisplay, display and express, or comprise at least a portion of thediversity of the antibody family, the vectors or genetic packages beingcharacterized by variegated DNA sequences that encode a heavy chain CDR3selected from the group consisting of:

-   -   (1) YYCA21111YFDYWG, wherein 1 is an equimolar mixture of each        amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,        T, V, W and Y; and 2 is an equimolar mixture of K and R;    -   (2) YYCA2111111YFDYWG, wherein 1 is an equimolar mixture of each        amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,        T, V, W and Y; and 2 is an equimolar mixture of K and R;    -   (3) YYCA211111111YFDAYTG, wherein 1 is an equimolar mixture of        each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q,        R, S, T, V, W and Y; and 2 is an equimolar mixture of K and R;    -   (4) YYCAR111S2S3111YFDYWG, wherein 1 is an equimolar mixture of        each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q,        R, S, T, V, W and Y; and 2 is an equimolar mixture of S and G;        and 3 is an equimolar mixture of Y and W;    -   (5) YYCA2111CSG11CY1YFDYWG, wherein 1 is an equimolar mixture of        each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q,        R, S, T, V, W and Y; and 2 is an equimolar mixture of K and R;    -   (6) YYCA211S1TIFG11111YFDYWG, wherein 1 is an equimolar mixture        of each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P,        Q, R, S, T, V, W and Y; and 2 is an equimolar mixture of K and        R;    -   (7) YYCAR111YY2S3344111YFDYWG, wherein 1 is an equimolar mixture        of each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P,        Q, R, S, T, V, W and Y; 2 is an equimolar mixture of D and S;        and 3 is an equimolar mixture of S and G;    -   (8) YYCAR1111YC2231CY111YFDYWG, wherein 1 is an equimolar        mixture of each amino acid residues A, D, E, F, G, H, I, K, L,        M, N, P, Q, R, S, T, V, W and Y; 2 is an equimolar mixture of S        and G; and 3 is an equimolar mixture of T, D and G; and    -   (9) mixtures of vectors or genetic packages characterized by any        of the above DNA sequences, preferably the HC CDR3s (1)        through (8) are in the following proportions in the mixture:    -   (1) 0.10    -   (2) 0.14    -   (3) 0.25    -   (4) 0.13    -   (5) 0.13    -   (6) 0.11

(7) 0.04 and

-   -   (8) 0.10; and more preferably the HC CDR3s (1) through (8) are        in the following proportions in the mixture:    -   (1) 0.02    -   (2) 0.14    -   (3) 0.25    -   (4) 0.14    -   (5) 0.14    -   (6) 0.12    -   (7) 0.08 and    -   (8) 0.11.

Preferably, 1 in one or all of HC CDR3s (1) through (8) is 0.095 of eachof G and Y and 0.048 of each of A, D, E, F, H, I, K, L, M, N, P, Q, R,S, T, V, and W.

4. A focused library of vectors or genetic packages that display,display and express, or comprise a member of a diverse family of humanantibody related peptides, polypeptides and proteins and collectivelydisplay, display and express, or comprise at least a portion of thediversity of the antibody family, the vectors or genetic packages beingcharacterized by variegated DNA sequences that encodes a kappa lightchain CDR1 selected from the group consisting of:

-   -   (1) RASQ<1>V<2><2><3>LA    -   (2) RASQ<1>V<2><2><2><3>LA;        wherein <1> is an equimolar mixture of amino acid residues        ADEFGHIKLMNPQRSTVWY; <2> is 0.2 S and 0.044 of each of        ADEFGHIKLMNPQRTVWY; and <3> is 0.2Y and 0.044 each of        ADEFGHIKLMNPQRTVW and Y; and    -   (3) mixtures of vectors or genetic packages characterized by any        of the above DNA sequences, preferably in the ratio CDR1s        (1):(2)::0.68:0.32.

5. A focused library of vectors or genetic packages that display,display and express, or comprise a member of a diverse family of humanantibody related peptides, polypeptides and proteins and collectivelydisplay, display and express, or comprise at least a portion of thediversity of the antibody family, the vectors or genetic packages beingcharacterized by variegated DNA sequences that encode a kappa lightchain CDR2 having the sequence:

-   -   <1>AS<2>R<4><1>,        wherein <1> is an equimolar mixture of amino acid residues        ADEFGHIKLMNPQRSTVWY; <2> is 0.2 S and 0.044 of each of        ADEFGHIKLMNPQRTVWY; and <4> is 0.2 A and) 0.044 each of        DEFGHIKLMNPQRSTVWY.

6. A focused library of vectors or genetic packages that display,display and express, or comprise a member of a diverse family of humanantibody related peptides, polypeptides and proteins and collectivelydisplay, display and express, or comprise at least a portion of thediversity of the antibody family, the vectors or genetic packages beingcharacterized by variegated DNA sequences that encode a kappa lightchain CDR3 selected from the groups consisting of:

-   -   (1) QQ<3><1><1><1>P<1>T,        wherein <1> is an equimolar mixture of amino acid residues        ADEFGHIKLMNPQRSTVWY; <3> is 0.2 Y and 0.044 each of        ADEFGHIKLMNPQRTVW;    -   (2) QQ33111P, wherein 1 and 3 are as defined in (1) above;    -   (3) QQ3211PP1T, wherein 1 and 3 are as defined in (1) above and        2 is 0.2 S and 0.044 each of ADEFGHIKLMNPQRTVWY; and    -   (4) mixtures of vectors or genetic packages characterized by any        of the above DNA sequences, preferably in the ratio CDR3s        (1):(2):(3)::0.65:0.1:0.25.

7. A focused library of vectors or genetic packages that display,display and express, or comprise a member of a diverse family of humanantibody related peptides, polypeptides and proteins and collectivelydisplay, display and express, or comprise at least a portion of thediversity of the antibody family, the vectors or genetic packages beingcharacterized by variegated DNA sequences that encode a lambda lightchain CDR1 selected from the group consisting of:

-   -   (1) TG<1>SS<2>VG<1><3><2><3>VS,        wherein <1> is 0.27 T, 0.27 G and 0.027 each of        ADEFHIKLMNPQRSVWY, <2> is 0.27 D, 0.27 N and 0.027 each of        AEFGHIKLMPQRSTVWY, and <3> is 0.36 Y and 0.036 each of        ADEFGHIKLMNPQRSTVW;    -   (2) G<2><4>L<4><4><4><3><4><4>,        wherein <2> is as defined in (1) above and <4> is an equimolar        mixture of amino acid residues ADEFGHIKLMNPQRSTVWY; and    -   (3) mixtures of vectors or genetic packages characterized by any        of the above DNA sequences, preferably in the ratio CDR1s        (1):(2)::0.67:0.33.

8. A focused library of vectors or genetic packages that display,display and express, or comprise a member of a diverse family of humanantibody related peptides, polypeptides and proteins and collectivelydisplay, display and express, or comprise at least a portion of thediversity of the antibody family, the vectors or genetic packages beingcharacterized by variegated DNA sequences that encode a lambda lightchain CDR2 has the sequence:

-   -   <4><4><4><2>RPS,        wherein <2> is 0.27 D, 0.27 N, and 0.027 each of        AEFGHIKLMPQRSTVWY and <4> is an equimolar mixture of amino acid        residues ADEFGHIKLMNPQRSTVW.

9. A focused library of vectors or genetic packages that display,display and express, or comprise a member of a diverse family of humanantibody related peptides, polypeptides and proteins and collectivelydisplay, display and express, or comprise at least a portion of thediversity of the antibody family, the vectors or genetic packages beingcharacterized by variegated DNA sequences that encode a lambda lightchain CDR3 selected from the group consisting of:

-   -   (1) <4><5><4><2><4>S<4><4><4><4>V,        wherein <2> is 0.27 D, 0.27 N, and 0.027 each of        AEFGHIKLMPQRSTVWY; <4> is an equimolar mixture of amino acid        residues ADEFGHIKLMNPQRSTVW; and <5> is 0.36 S and 0.0355 each        of ADEFGHIKLMNPQRTVWY;    -   (2) <5>SY<1><5>S<5><1><4>V, wherein <1> is an equimolar mixture        of ADEFGHIKLMNPQRSTVWY; and <4> and <5> are as defined in (1)        above; and    -   (3) mixtures of vectors or genetic packages characterized by any        of the above DNA sequences, preferably in the ratio CDR3s        (1):(2)::1:1.

10. A focused library comprising variegated DNA sequences that encode aheavy chain CDR selected from the group consisting of:

-   -   (1) one or more of the heavy chain CDR1s of paragraph 1 above;    -   (2) one or more of the heavy chain CDR2s of paragraph 2 above;    -   (3) one or more of the heavy chain CDR3s of paragraph 3 above;        and    -   (4) mixtures of vectors or genetic packages characterized by        (1), (2) and (3).

11. The focused library comprising one or more of the variegated DNAsequences that encodes a heavy chain CDR of paragraphs 1, 2 and 3 andfurther comprising variegated DNA sequences that encodes a light chainCDR selected from the group consisting of

-   -   (1) one or more the kappa light chain CDR1s of paragraph 4;    -   (2) the kappa light chain CDR2 of paragraph 5;    -   (3) one or more of the kappa light chain CDR3s of paragraph 6;    -   (4) one or more of the kappa light chain CDR1s of paragraph 7;    -   (5) the lambda light chain CDR2 of paragraph 8;    -   (6) one or more of the lambda light chain CDR3s of paragraph 9;        and    -   (7) mixtures of vectors and genetic packages characterized by        one or more of (1) through (6).

12. A population of variegated DNA sequences as described in paragraphs1-11 above.

13. A population of vectors comprising the variegated DNA sequences asdescribed in paragraphs 1-11 above.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Antibodies (“Ab”) concentrate their diversity into those regions thatare involved in determining affinity and specificity of the Ab forparticular targets. These regions may be diverse in sequence or inlength. Generally, they are diverse in both ways. However, withinfamilies of human antibodies the diversities, both in sequence and inlength, are not truly random. Rather, some amino acid residues arepreferred at certain positions of the CDRs and some CDR lengths arepreferred. These preferred diversities account for the natural diversityof the antibody family.

According to this invention, and as more fully described below,libraries of vectors and genetic packages that more closely mirror thenatural diversity, both in sequence and in length, of antibody families,or portions thereof are prepared and used.

Human Antibody Heavy Chain Sequence and Length Diversity

(a) Framework

The heavy chain (“HC”) Germ-Line Gene (GLG) 3-23 (also known as VP-47)accounts for about 12% of all human Abs and is preferred as theframework in the preferred embodiment of the invention. It should,however, be understood that other well-known frameworks, such as 4-34,3-30, 3-30.3 and 4-30.1, may also be used without departing from theprinciples of the focused diversities of this invention.

In addition, JH4 (YFDYWGQGTLVTUSS) occurs more often than JH3 in nativeantibodies. Hence, it is preferred for the focused libraries of thisinvention. However, JH3 (AFDIWGQGTMVTVSS) could as well be used.

(b) Focused Length Diversity: CDR1, 2 and 3

(i) CDR1

For CDR1, GLGs provide CDR1s only of the lengths 5, 6, and 7. Mutationsduring the maturation of the V-domain gene, however, can lead to CDR1shaving lengths as short as 2 and as long as 16. Nevertheless, length 5predominates. Accordingly, in the preferred embodiment of thisinvention, the preferred HC CDR1 is 5 amino acids, with less preferredCDR is having lengths of 7 and 14. In the most preferred libraries ofthis invention, all three lengths are used in proportions similar tothose found in natural antibodies.

(ii) CDR2

GLGs provide CDR2s only of the lengths 15-19, but mutations duringmaturation may result in CDR2s of lengths from 16 to 28 amino acids. Thelengths 16 and 17 predominate in mature Ab genes. Accordingly, length 17is the preferred length for HC CDR2 of the present invention. Lesspreferred HC CDR2s of this invention have lengths 16 and 19. In the mostpreferred focused libraries of this invention, all three lengths areincluded in proportions similar to those found in natural antibodyfamilies.

(iii) CDR3

HC CDR3s vary in length. About half of human HCs consist of thecomponents: V::nz::D::ny::JHn where V is a V gene, nz is a series ofbases (mean 12) that are essentially random, D is a D segment, oftenwith heavy editing at both ends, ny is a series of bases (mean 6) thatare essentially random, and JH is one of the six JH segments, often withheavy editing at the 5′ end. The D segments appear to provide spacersegments that allow folding of the IgG. The greatest diversity is at thejunctions of V with D and of D with JH.

In the preferred libraries of this invention both types of HC CDR3s areused. In HC CDR3s that have no identifiable D segment, the structure isV::nz::JHn where JH is usually edited at the 51 end. In HC CDR3s thathave an identifiable D segment, the structure is V::nz::D::ny::JHn.

(c) Focused Sequence Diversity: CDR1, 2 and 3

(i) CDR1

In 5 amino acid length CDR1, examination of a 3D model of a humanized Abshowed that the side groups of residues 1, 3, and 5 were directed towardthe combining pocket. Consequently, in the focused libraries of thisinvention, each of these positions may be selected from any of thenative amino acid residues, except cysteine (“C”). Cysteine can formdisulfide bonds, which are an important component of the canonical Igfold. Having free thiol groups could, thus, interfere with properfolding of the HC and could lead to problems in production ormanipulation of selected Abs. Thus, in the focused libraries of thisinvention cysteine is excluded from positions 1, 3 and 5 of thepreferred 5 amino acid CDR1s. The other 19 natural amino acids residuesmay be used at positions 1, 3 and 5. Preferably, each is present inequimolar ratios in the variegated libraries of this invention.

3D modeling also suggests that the side groups of residue 2 in a 5 aminoacid CDR1 are directed away from the combining-pocket. Although thisposition shows substantial diversity, both in GLG and mature genes, inthe focused libraries of this invention this residue is preferably Tyr(Y) because it occurs in 681/820 mature antibody genes. However, any ofthe other native amino acid residues, except Cys (C), could also be usedat this position.

For position 4, there is also some diversity in GLG and mature antibodygenes. However, almost all mature genes have uncharged hydrophobic aminoacid residues: A, G, L, P, F, M, W, I, V, at this position. Inspectionof a 3D model also shows that the side group of residue 4 is packed intothe innards of the HC. Thus, in the preferred embodiment of thisinvention which uses framework 3-23, residue 4 is preferably Met becauseit is likely to fit very well into the framework of 3-23. With otherframeworks, a similar fit consideration is used to assign residue 4.

Thus, the most preferred HC CDR1 of this invention consists of the aminoacid sequence <1>Y<1>M<1>where <1> can be any one of amino acidresidues: A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,. V, W, Y (notC), preferably present at each position in an equimolar amount. Thisdiversity is shown in the context of a framework 3-23:JH4 in Table 1. Ithas a diversity of 6859-fold.

The two less preferred HC CDR1s of this invention have length 7 andlength 14. For length 7, a preferred variegation is(S/T)₁(S/G/<1>)₂(S/G/<1>)₃Y₄Y₅W₆(S/G/<1>)₇; where (S/T) indicates anequimolar mixture of Ser and Thr codons; (S/G/<1>) indicates a mixtureof 0.2025 S, 0.2025 G, and 0.035 for each of A, D, E, F, H, I, K, L, M,N, P, Q, R, T, V, W, Y. This design gives a predominance of Ser and Glyat positions 2, 3, and 7, as occurs in mature HC genes. For length 14, apreferred variegation is VSGGSIS<1><1><1>YYW<1>, where <1> is anequimolar mixture of the 19 native amino acid residues, except Cys (C).

The DNA that encodes these preferred HC CDR1s is preferably synthesizedusing trinucleotide building blocks so that each amino acid residue ispresent in essentially equimolar or other described amounts. Thepreferred codons for the <1> amino acid residues are gct, gat, gag, ttt,ggt, cat, att, aag, ctt, atg, aat, cct, cag, cgt, tct, act, gtt, tgg,and tat. Of course, other codons for the chosen amino acid residue couldalso be used.

The diversity oligonucleotide (ON) is preferably synthesized from BspEIto BstXI (as shown in Table 1) and can, therefore, be incorporatedeither by PCR synthesis using overlapping ONs or introduced by ligationof BspEI/BstXI-cut fragments. Table 2 shows the oligonucleotides thatembody the specified variegations of the preferred length 5 HC CDR1s ofthis invention. PCR using ON-R1V1vg, ON-R1top, and ON-R1bot gives adsDNA product of 73 base pairs, cleavage with BspEI and BstXI trims 11and 13 bases from the ends and provides cohesive ends that can beligated to similarly cut vector having the 3-23 domain shown in Table 1.Replacement of ON-R1V1vg with either ONR1V2vg or ONR1V3vg (see Table 2)allows synthesis of the two alternative diversity patterns—the 7 residuelength and the 14 residue length HC CDR1.

The more preferred libraries of this invention comprise the 3 preferredHC CDR1 length diversities. Most preferably, the 3 lengths should beincorporated in approximately the ratios in which they are observed inantibodies selected without reference to the length of the CDRs. Forexample, one sample of 1095 HC genes have the three lengths present inthe ratio: L=5:L=7:L=14::820:175:23::0.80:0.17:0.02. This is thepreferred ratio in accordance with this invention.

(ii) CDR2

Diversity in HC CDR2 was designed with the same considerations as for HCCDR1: GLG sequences, mature sequences and 3D structure. A preferredlength for CDR2 is 17, as shown in Table 1. For this preferred 17 lengthCDR2, the preferred variegation in accordance with the invention is:<2>I<2><3>SGG<1>T<1>YADSVKG, where <2>indicates any amino acid residueselected from the group of Y, R, W, V, G and S (equimolar mixture), <3>is P, S and G or P and S only (equimolar mixture), and <1> is any nativeamino acid residue except C (equimolar mixture).

ON-R2V1vg shown in Table 3 embodies this diversity pattern. It ispreferably synthesized so that fragments of dsDNA containing the BstXIand XbaI site can be generated by PCR. PCR with ON-R2V1vg, ON-R2top, andONR2bot gives a dsDNA product of 122 base pairs. Cleavage with BstXI andXbaI removes about 10 bases from each end and produces cohesive endsthat can be ligated to similarly cut vector that contains the 3-23geneshown in Table 1.

In an alternative embodiment for a 17 length HC CDR2, the followingvariegation may be used: <1>I<4><1><1>G<5><1><1><1>YADSVKG, where <1> isas described above for the more preferred alternative of HC CDR2; <4>indicates an equimolar mixture of DINSWY, and <5> indicates an equimolarmixture of SGDN. This diversity pattern is embodied in ON-R2V2vg shownin Table 3. Preferably, the two embodiments are used in equimolarmixtures in the libraries of this invention.

Other preferred HC CDR2s have lengths 16 and 19. Length 16:<1>I<4><1><1>G<5<1><1>YNPSLKG; Length 19:<1>I<8>S<1><1><1>GGYY<1>YAASVKG, wherein <1> is an equimolar mixture ofall native amino acid residues except C; <4> is a equimolar mixture ofDINSWY; <5> is an equimolar mixture of SGDN; and <8> is 0.27 R and 0.027of each of residues ADEFGHIKLMNPQSTVWY. Table 3 shows ON-R2V3vg whichembodies a preferred CDR2 variegation of length 16 and ON-R2V4vg whichembodies a preferred CDR2 variegation of length 19. To prepare thesevariegations ON-R2V3vg may be PCR amplified with ON-R2top and ON-R2bo3and ON-R2V4vg may be PCR amplified with ON-R2top and ON-R2-bo4. SeeTable 3. In the most preferred embodiment of this invention, all threeHC CDR2 lengths are used. Preferably, they are present in a ratio17:16:19::579:464:31::0.54:0.43:0.03.

(iii) CDR3

The preferred libraries of this invention comprise several HC CDR3components. Some of these will have only sequence diversity. Others willhave sequence diversity with embedded D segments to extend the length,while also incorporating sequences known to allow Igs to fold. The HCCDR3 components of the preferred libraries of this invention and theirdiversities are depicted in Table 4: Components 1-8.

This set of components was chosen after studying the sequences of 1383human HC sequences. The proposed components are meant to fulfill thefollowing goals:

1) approximately the same distribution of lengths as seen in native Abgenes;

2) high level of sequence diversity at places having high diversity innative Ab genes; and

3) incorporation of constant sequences often seen in native Ab genes.

Component 1 represents all the genes having lengths 0 to 8 (countingfrom the YYCAR motif at the end of FR3 to the WG dipeptide motif nearthe start of the J region, i.e., FR4). Component 2 corresponds the allthe genes having lengths 9 or 10. Component 3 corresponds to the geneshaving lengths 11 or 12 plus half the genes having length 13. Component4 corresponds to those having length 14 plus half those having length13. Component 5 corresponds to the genes having length 15 and half ofthose having length 16. Component 6 corresponds to genes of length 17plus half of those with length 16. Component 7 corresponds to those withlength 18. Component 8 corresponds to those having length 19 andgreater. See Table 4.

For each HC CDR3 residue having the diversity <1>, equimolar ratios arepreferably not used. Rather, the following ratios are used 0.095 [G andY] and 0.048 [A, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, and W].Thus, there is a double dose of G and Y with the other residues being inequimolar ratios. For the other diversities, e.g., KR or SG, theresidues are present in equimolar mixtures.

In the preferred libraries of this invention the eight components arepresent in the following fractions: 1 (0.10), 2 (0.14), 3 (0.25), 4(0.13), 5 (0.13), 6 (0.11), 7 (0.04) and 8 (0.10). See Table 4.

In the more preferred embodiment of this invention, the amounts of theeight components is adjusted because the first component is not complexenough to justify including it as 10% of the library. For example, ifthe final library were to have 1×10⁹ members, then 1×10⁸ sequences wouldcome from component 1, but it has only 2.6×10⁵ CDR3 sequences so thateach one would occur in ˜385 CDR1/2 contexts. Therefore, the morepreferred amounts of the eight components are 1(0.02), 2(0.14), 3(0.25),4(0.14), 5(0.14), 6(0.12), 7(0.08), 8(0.11). In accordance with the morepreferred embodiment component 1 occurs in ˜77 CDR1/2 contexts and theother, longer CDR3s occur more often.

Table 5 shows vgDNA that embodies each of the eight HC CDR3 componentsshown in Table 4. In Table 5, the oligonucleotides (ON) Ctop25, CtprmA,CBprmB, and CBot25 allow PCR amplification of each of the variegated ONs(vgDNA): C1t08, C2t10, C3t12, C4t14, C5t15, C6t17, C7t18, and C8t19.After amplification, the dsDNA can be cleaved with AflII and BstEII (orKpnI) and ligated to similarly cleaved vector that contains theremainder of the 3-23 domain. Preferably, this vector already containsdiversity in one, or both, of CDR1 and CDR2 as disclosed herein. Mostpreferably, it contains diversity in both the CDR1 and CDR2 regions. Itis, of course, to be understood that the various diversities can beincorporated into the vector in any order.

Preferably, the recipient vector originally contains a stuffer in placeof CDR1, CDR2 and CDR3 so that there will be no parental sequence thatwould then occur in the resulting library. Table 6 shows a version ofthe V3-23 gene segment with each CDR replaced by a short segment thatcontains both stop codons and restriction sites that will allow specificcleavage of any vector that does not have the stuffer removed. Thestuffer can either be short and contain a restriction enzyme site thatwill not occur in the finished library, allowing removal of vectors thatare not cleaved by both AflII and BstEII (or KpnI) and religated.Alternatively, the stuffer could be 200-400 bases long so that uncleavedor once cleaved vector can be readily separated from doubly cleavedvector.

Human Antibody Light Chain: Sequence and Length Diversity

(i) Kappa Chain

(a) Framework

In the preferred embodiment of this invention, the kappa light chain isbuilt in an A27 framework with a JK1 region. These are the most common Vand J regions in the native genes. Other frameworks, such as 012, L2,and A11, and other J regions, such as JK4, however, may be used withoutdeparting from the scope of this invention.

(b) CDR1

In native human kappa chains, CDR1s with lengths of 11, 12, 13, 16, and17 were observed with length 11 being predominant and length 12 beingwell represented. Thus, in the preferred embodiments of this inventionLC CDR1s of length 11 and 12 are used in an and mixture similar to thatobserved in native antibodies), length 11 being most preferred. Length11 has the following sequence: RASQ<1>V<2><2><3>LA and Length 12 has thefollowing sequence: RASQ<1>V<2><2><2><3>LA, wherein <1> is an equimolarmixture of all of the native amino acid residues, except C, <2> is 0.2 Sand 0.044 of each of ADEFGHIKLMNPQRTVWY, and <3> is 0.2 Y and 0.044 eachof A, D, E, F, G, H, I, K, L, M, N, P, Q, R, T, V, W and Y. In the mostpreferred embodiment of this invention, both CDR1 lengths are used.Preferably, they are present in a ratio of 11:12::154:73::0.68:0.32.

(c) CDR2

In native kappa, CDR2 exhibits only length 7. This length is used in thepreferred embodiments of this invention. It has the sequence<1>AS<2>R<4><1>, wherein <1> is an equimolar mixture of amino acidresidues ADEFGHIKLMNPQRSTVWY; <2> is 0.2 S and 0.004 of each ofADEFGHIKLMNPQRTVWY; and <4> is 0.2 A and 0.044 of each ofDEFGHIKLMNPQRSTUWY.

(d) CDR3

In native kappa, CDR3 exhibits lengths of 1, 4, 6, 7, 8, 9, 10, 11, 12,13, and 19. While any of these lengths and mixtures of them can beemployed in this invention, we prefer lengths 8, 9 and 10, length 9being more preferred. For the preferred Length 9, the sequence isQQ<3><1><1><1>P<1>T, wherein <1> is an equimolar mixture of amino acidresidues ADEFGHIKLMNPQRSTVWY and <3> is 0.2 Y and 0.044 each ofADEFGHIKLMNPQRSVW. Length 8 is preferably QQ33111P and Length 10 ispreferably QQ3211PP1T, wherein 1 and 3 are as defined for Length 9 and 2is S (0.2) and 0.044 each of ADEFGHIKLMNPQRTVWY. A mixture of all 3lengths being most preferred (ratios as in native antibodies), i.e.,8:9:10::28:166:63::0.1:0.65:0.25.

Table 7 shows a kappa chain gene of this invention, including a PlacZpromoter, a ribosome-binding site, and signal sequence (M13 III signal).The DNA sequence encodes the GLG amino acid sequence, but does notcomprise the GLG DNA sequence. Restriction sites are designed to fallwithin each framework region so that diversity can be cloned into theCDRs. XmaI and EspI are in FR1, SexAI is in FR2, RsrII is in FR3, andKpnI (or Acc65I) are in FR4. Additional sites are provided in theconstant kappa chain to facilitate construction of the gene.

Table 7 also shows a suitable scheme of variegation for kappa. In CDR1,the most preferred length 11 is depicted. However, most preferably bothlengths 11 and 12 are used. Length 12 in CDR1 can be construed byintroducing codon 51 as <2> (i.e. a Ser-biased mixture). CDR2 of kappais always 7 codons. Table 7 shows a preferred variegation scheme forCDR2. Table 7 shows a variegation scheme for the most preferred CDR3(length 9). Similar variegations can be used for CDRs of length 8 and10. In the preferred embodiment of this invention, those three lengths(8, 9 and 10) are included in the libraries of this invention in thenative ratios, as described above.

Table 9 shows series of diversity oligonucleotides and primers that maybe used to construct the kappa chain diversities depicted in Table 7.

(ii) Lambda Chain

(a) Framework

The lambda chain is preferably built in a 2a2 framework with an L2Jregion. These are the most common V and J regions in the native genes.Other frameworks, such as 31, 4b, 1a and 6a, and other J regions, suchas L1J, L3J and L7J, however, may be used without departing from thescope of this invention.

(b) CDR1

In native human lambda chains, CDR1s with length 14 predominate, lengths11, 12 and 13 also occur. While any of these can be used in thisinvention, lengths 11 and 14 are preferred. For length 11 the sequenceis: TG<2><4>L<4><4><4><3><4><4>and for Length 14 the sequence is:TG<1>5S<2>VG<1><3><2><3>VS, wherein <1> is 0.27 T, 0.27 G and 0.027 eachof ADEFHIKLMNPQRSVWY; <2> is 0.27 D, 0.27 N and 0.027 each ofAEFGHIKLMPQRSTVWY; <3> is 0.36 Y and 0.0355 each of ADEFGHIKLMNPQRSTVW;and <4> is an equimolar mixture of amino acid residuesADEFGHIKLMNPQRSTVWY. Most preferably, mixtures (similar to thoseoccurring in native antibodies) preferably, the ratio is11:14::23:46::0.33: 0.67 of the three lengths are used.

(c) CDR2

In native human lambda chains, CDR2s with length 7 are by far the mostcommon. This length is preferred in this invention. The sequence of thisLength 7 CDR2 is <4><4><4><2>RPS, wherein <2> is 0.27 D, 0.27 N, and0.027 each of AEFGHIKLMPQRSTVWY and <4> is an equimolar mixture of aminoacid residues ADEFGHIKLMNPQRSTVW.

(d) CDR3

In native human lambda chains, CDR3s of length 10 and 11 predominate,while length 9 is also common. Any of these three lengths can be used inthe invention. Length 11 is preferred and mixtures of 10 and 11 morepreferred. The sequence of Length 11 is <4><5><4><2><4>S<4><4><4><4>V,where <2> and <4> are as defined for the lambda CDR1 and <5> is 0.36 Sand 0.0355 each of ADFFGHIKLMNPQRTVWY. The sequence of Length 10 is<5>SY<1><5>S<5><1><4>V, wherein <1> is an equimolar mixture ofADEFGHIKLMNPQRSTVWY; and <4> and <5> are as defined for Length 11. Thepreferred mixtures of this invention comprise an equimolar mixture ofLength 10 and Length 11. Table 8 shows a preferred focused lambda lightchain diversity in accordance with this invention.

Table 9 shows a series of diversity oligonucleotides and primers thatmay be used to construct the lambda chain diversities depicted in Table7.

Method of Construction of the Genetic Package

The diversities of heavy chain and the kappa and lambda light chains arebest constructed in separate vectors. First a synthetic gene is designedto embody each of the synthetic variable domains. The light chains arebounded by restriction sites for ApaLI (positioned at the very end ofthe signal sequence) and AscI (positioned afer the stop codon). Theheavy chain is bounded by SfiI (positioned within the Pe1B signalsequence) and NotI (positioned in the linker between CH1 and the anchorprotein). Signal sequences other than Pe1B may also need, e.g., a M13pIII signal sequence.

The initial genes are made with “stuffer” sequences in place of thedesired CDRs. A “Stuffer” is a sequence that is to be cut away andreplaced by diverse DNA but which does not allow expression of afunctional antibody gene. For example, the stuffer may contain severalstop codons and restriction sites that will not occur in the correctfinished library vector. For example, in Table 10, the stuffer for CDR1of kappa A27 contains a StuI site. The vgDNA for CDR1 is introduced as acassette from EspI, XmaI, or AflII to either SexAI or KasI. After theligation, the DNA is cleaved with StuI; there should be no StuI sites inthe desired vectors.

The sequences of the heavy chain gene with stuffers is depicted in Table6. The sequences of the kappa light chain gene with stuffers is depictedin Table 10. The sequence of the lambda light chain gene with stuffersis depicted in Table 11.

In another embodiment of the present intention the diversities of heavychain and the kappa or lambda light chains are constructed in a singlevector or genetic packages (e.g., for display or display and expression)having appropriate restriction sites that allow cloning of these chains.The processes to construct such vectors are well known and widely usedin the art. Preferably, a heavy chain and Kappa light chain library anda heavy chain and lambda light chain library would be preparedseparately. The two libraries, most preferably, will then be mixed inequimolar amounts to attain maximum diversity.

Most preferably, the display is had on the surface of a derivative ofM13 phage. The most preferred vector contains all the genes of M13, anantibiotic resistance gene, and the display cassette. The preferredvector is provided with restriction sites that allow introduction andexcision of members of the diverse family of genes, as cassettes. Thepreferred vector is stable against rearrangement under the growthconditions used to amplify phage.

In another embodiment of this invention, the diversity captured by themethods of the present invention may be displayed and/or expressed in aphagemid vector (e.g., pCES1) that displays and/or expresses thepeptide, polypeptide or protein. Such vectors may also be used to storethe diversity for subsequent display and/or expression using othervectors or phage.

In another embodiment of this invention, the diversity captured by themethods of the present invention may be displayed and/or expressed in ayeast vector. TABLE 1 3-23:JH4 CDR1/2 diversity = 1.78 × 10⁸                                FR1 (VP47/V3-23).......           20 2122              23  24  25  26  27  28  29  30           A  M  A               E   V   Q   L   L   E   S   Gctgtctgaac cc atg gcc           gaa|gtt|caa|ttg|tta|gag|tct|ggt|Scab...... NcoI....                      MfeI-------------FR1---------------------------------31  32  33  34  35  36  37  38  39  40  41  42  43  44  45 G   G   L   V   Q   P   G   G   S   L   R   L   S   C   A|ggc|ggt|cct|gtt|cag|cct|ggt|ggt|tct|tta|cgt|ctt|tct|tgc|gtc|     Sitesof variegation    <1>        <1> <1> <1>            6859-fold diversity----FR1------------------->|.....CDR1..................|---FR2------46  47  48  49  50  51  52  53  54  55  56  57  58  59  60 A   S   G   F   T   F   S   -  Y  -   M   -   W  V  R|gct|tcc|gga|ttc|act|ttc|tct|-|tac|-|atg|-|tgg|gtt|cgc|      BspEI                   BsiWI                   BstXI.                      Sites of variegation-><2>    <2> <3>-------FR2-------------------------------->|...CDR2..........61  62  63  64  65  66  67  68  69  70  71  72  73  74  75 Q   A   P   G   K   G   L   E   W   V   S   -   I   -   -|caa|gct|cct|ggt|aaa|ggt|ttg|gag|tgg|gtt|tct| - |atc| - | - | ...BstXI            <1>     <1>  25992-fold diversity in CDR2....CDR2............................................|---FR3--- 76  77  78  79  80  81  82  83  84  85  86  87  88  89  90  S   G   G   -   T   -   Y   A   D   S   V   K   G   R   F|tct|ggt|ggc| - |act| - |tat|gct|gac|tcc|gtt|aaa|ggt|cgc|ttc|-------FR3--------------------------------------------------- 91  92  93  94  95  96  97  98  99  100 101 102 103 104 105 T   I   S   R   D   N   S   K   N   T   L   Y   L   Q   M|act|atc|tct|aga|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|         XbaI---FR3---------------------------------------------------->|  106 107108 109 110 111 112 113 114 115 116 117 118 119 120  N   S   L   R   A   E   D   T   A   V   Y   Y   C   A   K|aac|agc|tta|agg|gct|gag|tac|acc|gct|gtc|tac|tac|tgc|gcc|aaa|        AflII .......CDR3.................| Replaced by the variouscomponents!  121 122 123 124 125 126 127   D   Y   E   G   T   G   Y|gac|tat|gaa|ggt|act|ggt|tat||------FRV----(JH4)------------------------------------------  Y   F   D   Y   W   G   Q   G   T   L   V   T   V   S   S|tat|ttc|gat|tat|tgg|ggt|caa|ggt|acc|ctg|gtc|acc|gtc|tct|agt|...                             KpnI       BstEII<1> = Codons for ADEFGHIKLMNPQRSTVWY (equimolar mixture)<2> = Codons for YRWVGS (equimolar mixture)<3> = Codons for PS or PS and G (equimolar mixture)

TABLE 2 OligoNucleotides used to variegate CDR1 of human HC CDR1 - 5residues (ON-R1V1vg):5′ct|tcc|gga|ttc|act|ttc|tct|<1>|tac|<1>|atg|<1>|tgg|gtt|cgc|caa|gct|cct|gg|-3′(ON-R1top): 5′-cctactgtct|tcc|gga|ttc|act|ttc|tct-3′ (ON-R1bot) [RC]:5′-tgg|gtt|cgc|caa|gct|cct|ggttgctcactc-3′ CDR1 - 7 residues(ON-R1V2vg):5′-ct|tcc|gga|ttc|act|ttc|tct|<6>|<7>|<7>|tac|tac|tgg|<7>|tgg|gtt|cgc|caa|gct|cct|gg-3′ CDR1 - 14 residues (ON-R1V3vg):5′-ct|tcc|gga|ttc|act|ttc|tct|atc|agc|ggt|ggt|tct|atc|tcc|<1>|<1>|<1>|-tac|tac|tgg|<1>|tgg|gtt|cgc|caa|gct|cct|gg-3′<1> = Codons of ADEFGHIKLMNPQRSTVWY 1:1<6> = Codons for ST, 1:1<7> = 0.2025 (Codons for SG) + 0.035 (Codons for ADEFHIKLMNPQRTVWY)<1> = Codons for ADEFGHIKLMNPQRSTVWY 1:1

TABLE 3 Oligonucleotides used to variegate CDR2 of human HC CDR2 - 17residues (ON-R2V1vg):5′-ggt|ttg|gag|tgg|gtt|tct|<2>|atc|<2>|<3>|tct|ggt|ggc|<1>|act|<1>|tat|gct|-gac|tcc|gtt|aaa|gg-3′ (ON-R2top):5′-ct|tgg|gtt|cgc|caa|gct|cct|ggt|aaa|ggt|ttg|gag|tgg|gtt|tct-3′(ON-R2bot) [RC]:5′-tat|gct|gac|tcc|gtt|aaa|ggt|cgc|ttc|act|atc|tct|aga|ttcctgtcac-3′(ON-R2V2vg):5′-ggt|ttg|gag|tgg|gtt|tct|<1>|atc|<4>|<1>|<1>|ggt|<5>|<1>|<1>|<1>|tat|gct|-gac|tcc|gtt|aaa|gg-3′ CDR2 - 16 residues (ON-R2V3vg):5′-ggt|ttg|gag|tgg|gtt|tct|<1>|atc|<4>|<1>|<1>|ggt|<5>|<1>|<1>|tat|aac|cct|tcc|ctt|aag|gg-3′ (ON-R2bo3) [RC]:5′-tat|aac|cct|tcc|ctt|aag|ggt|cgc|ttc|act|tct|aga|ttcctgtcac-3′ CDR2 -19 residues (ON-R2V4vg):5′-fft|ttg|gag|tgg|gtt|tct|<1>|atc|<8|agt|<1>|<1>|<1>|ggt|ggt|act|act|<1>|tat|gcc|gct|tcc|gtt|aag|gg-3′ (ON-R2bo4) [RC]:5′-tat|gcc|gct|tcc|gtt|aag|ggt|cgc|ttc|act|atc|tct|aga|ttcctgtcac-3′<1> = Codons for A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Wand Y (equimolar mixture)<2> = Codons for Y, R, W, V, G and S (equimolar mixture)<3> = Codons for P and S (equimolar mixture) or P, S and G (equimolarmixture)<4> =Codons for DINSWY (equimolar mixture)<5> =Codons for SGDN, (equimolar mixture)<1>, <2>, <3>, <4> and <5> are as defined above<8> is 0.27 R and 0.027 each of ADEFGHIKLMNPQSTVWY

TABLE 4 Preferred Components of HC CDR3 Preferred Fraction of AdjustedComponent Length Complexity Library Fraction 1 YYCA21111YFDYWG. 8 2.6× 10⁵ .10 .02 (1 = any amino acid residue, except C; 2 = K and R) 2YYCA2111111YFDYWG. 10 9.4 × 10⁷ .14 .14 (1 = any amino acid residue,except C; 2 = K and R 3 YYCA211111111YFDYTG. 12 3.4 × 10¹⁰ .25 .25 (1= any amino acid residue, except C; 2 = K and R 4 YYCAR111S2S3111YFDYWG.14 1.9 × 10⁸ .13 .14 (1 = any amino acid residue, except C; 2 = S and G3 = Y and W) 5 YYCA2111CSG11CY1YFDYWG. 15 9.4 × 10⁷ .13 .14 (1 = anyamino acid residue, except C; 2 = K and R 6 YYCA211S1TIFG11111YFDYWG. 171.7 × 10¹⁰ .11 .12 (1 = any amino acid residue, except C; 2 = K and R 7YYCAR111YY2S33YY111YFDYWG. 18 3.8 × 10⁸ .04 .08 (1 = any amino acidresidue, except C; 2 = D or G; 3 = S and G) 8YYCAR1111YC2231CY111YFDYWG. 19 2.0 × 10¹¹ .10 .11 (1 = any amino acidresidue, except C; 2 = S and G; 3 = T, D and G)

TABLE 5 Oligonucleotides used to variegate the eight components of HCCDR3 (Ctop25): 5′-gctctggtcaac|tta|agg|gct|gag|g-3′ (CtprmA):5′-gctctggtcaac|tta|agg|gct|gag|gac|acc|gct|gtc|tac|tac|tgc|gcc-3′              AflII... (CBprmB) [RC]:5′-|tac|ttc|gat|tac|tgg|ggc|caa|ggt|acc|ctg|gtc|acc|tcgctccacc-3′                                          BstEII... (CBot25) [RC]:5′-|ggt|acc|ctg|gtc|acc|tcgctccacc-3′ The 20 bases at 3′ end of CtprmAare identical to the most 5′ 20 bases of each of the vgDNA molecules.Ctop25 is identical to the most 5′ 25 bases of CtprmA. The 23 most3′ bases of CBprmB are the reverse complement of the most 3′ 23 bases ofeach of the vgDNA molecules. CBot25 is identical to the 25 bases at the5′ end of CBprmB. Component 1 (C1t08):5′-cc|gct|gtc|tac|tac|tgc|ggc|<2>|<1>|<1>|<1>|<1>|tac|ttc|gat|tac|tgg|ggc|caa|gg-3′Component 2 (C2t10):5′-cc|gct|gtc|tac|tac|tgc|gcc|<2>|<1>|<1>|<1>|<1>|<1>|<1>|tac|ttc|gat|tac|tgg|ggc|caa|gg-3′Component 3 (C3t12):5′-cc|gct|gtc|tac|tac|tgc|gcc|<2>|<1>|<1>|<1>|<1>|<1>|<1>|<1>|<1>|tac|ttc||gat|tac|-tgg|ggc|caa|gg-3′ Component 4 (C4t140):5′-cc|gct|gtc|tac|tac|tgc|gcc|cgt|<1>|<1>|<1>|tct|<2>|tct|<3>|<1>|<1>|<1>|tac|ttc|gat|-tac|tgg|ggc|caa|gg-3′ Component 5 (C5t15):5′-cc|gct|gtc|tac|tac|tgc|gcc|<2>|<1>|<1>|<1>|tgc|tct|ggt|<1>|<1>|tgc|tat|<1>|tac|-ttc|gat|tac|tgg|ggc|caa|gg-3′ Component 6 (C6t17):5′-cc|gct|gtc|tac|tac|tgc|gcc|<2>|<1>|<1>|tct<1>|act|atc|ttc|ggt|<1>|<1>|<1>|<1>|-<1>|tac|ttc|gat|tac|tgg|ggc|caa|gg-3′ Component 7 (C7t18):5′-cc|gct|gtc|tac|tac|tgc|ggc|cgt|<1>|<1>|<1>|tat|tac|<2>|tct|<3>|<3>|tac|tat|-<1>|<1>|<1>|tac|ttc|gat|tac|tgg|ggc|caa|gg-3′ Component 8 (c8t19):5′-cc|gct|gtc|tac|tac|tgc|gcc|cgt|<1>|<1>|<1>|<1>|tat|tgc|<2>|<2>|<3>|<1>|tgc|tat|-<1>|<1>|<1>|tac|ttc|gat|tac|tgg|ggc|caa|gg-3′<1> = 0.095 Y + 0.095 G + 0.048 each of the residues ADEFHIKLMNPQRSTVW,no C;<2> = K and R (equimolar mixture)<1> = 0.095 Y + 0.095 G + 0.048 each of ADEFHIKLMNPQRSTVW, no C;<2> = K and R (equimolar mixture)<1> = 0.095 Y + 0.095 G + 0.048 each of ADEFHIKLMNPQRSTVW, no C;<2> = K and R (equimolar mixture)<1> = 0.095 Y + 0.095 G + 0.048 each of ADEFHIKLMNPQRSTVW, no C;<2> = S and G (equimolar mixture);<3> = Y and W (equimolar mixture)<1> = 0.095 Y + 0.095 G + 0.048 each of ADEFHIKLMNPQRSTVW, no C;<2> = K and R (equimolar mixture)<1> = 0.095 Y + 0.095 G + 0.048 each of ADEFHIKLMNPQRSTVW, no C;<2> = K and R (equimolar mixture)<1> = 0.095 Y + 0.095 G + 0.048 each of ADEFHIKLMNPQRSTVW, no C;<2> = D and G (equimolar mixture);<3> = S and G (equimolar mixture)<1> = 0.095 Y + 0.095 G + 0.048 each of ADEFHIKLMNPQRSTVW, no C;<2> = S and G (equimolar mixture);<3> = TDG (equimolar mixture);

TABLE 6 3-23::JH4 Stuffers in place of CDRs                                 FR1 (DP47/V3-23)          20  21  22             23  24  25  26  27  28  29  30           A   M   A             E   V   Q   L   L   E   S   Gctgtctgaac cc atg gcc           gaa|gtt|caa|ttg|tta|gag|tct|ggt|Scab...... NcoI....                      MfeI--------------FR1-------------------------------------------- 31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  G   G   L   V   Q   P   G   G   S   L   R   L   S   C   A|ggc|ggt|ctt|gtt|cag|cct|ggt|ggt|tct|tta|cgt|ctt|tct|tgc|gct|----FR1-------------------->|...CDR1 stuffer....|---FR2------ 46  47  48  49  50  51  52  53  54  55  56  57  58  59  60  A   S   G   F   T   F   S   S   Y   A   |   |   W   V   R|gct|tcc|gaa|ttc|act|ttc|tct|tcg|tac|gct|tag|taa|tgg|gtt|cgc|      BspEI                     BsiWI                       BstXI. -------FR2-------------------------------->|...CDR2 stuffer. 61  62  63  64  65  66  67  68  69  70  71  72  73  74  75  Q   A   P   G   K   G   L   E   W   V   S   |   p   r   ||caa|gct|cct|ggt|aaa|ggt|ttg|gag|tgg|gtt|tct|taa|cct|agg|tag| ...BstXI                                        AvrII.. ...CDR2stuffer....................................|---FR3---  91  92  93  94  95  96  97  98  99 100 101 102 103 104 105  T   I   S   R   D   N   S   K   N   T   L   Y   L   Q   M|act|atc|tct|aga|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg|          XbaI ---FR3-----------..> CDR3 Stuffer------------->|  106 107108 109 110   N   S   L   R   A |aac|agc|tta|agg|gct|tag taa agg cct taa        AflII                StuI... |----- FR4---(JH4)------------------------------------------  Y   F   D   Y   W   G   Q   G   T   L   V   T   V   S   S|tat|ttc|gat|tat|tgg|ggt|caa|ggt|acc|ctg|gtc|acc|gtc|tct|agt|                              KpnI       BstEII

TABLE 7 A27:JH1 Human Kappa light chain gene gaggacc attgggccccctccgagact ctcgagcgca Scab...... Eco0109I           XhoI..           ApaI. acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacactttatgcttc       ..-35..          Plac                    ..-10.cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatgaccatgatta cgccaagctt tggagccttt tttttggaga ttttcaac   Pf1MI.......      Hind III M13 III signal sequence (AAse|)--------------------------> 1   2   3   4   5   6   7   8   9  10  11  12  13  14  15 M   K   K   L   L   F   A   I   P   L   V   V   P   F   Y gtg aag aagctc cta ttt gct atc ccg ctt gtc gtt ccg ttt tac --Signal-->FR1------------------------------------------> 16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  S   H   S   A   Q   S   V   L   T   Q   S   P   G   T   L|agc|cat|agt|gca|caa|tcc|gtc|ctt|act|caa|tct|cct|ggc|act|ctt|          ApaLI... ----- FR1------------------------------------->| CDR1------> 31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  S   L   S   P   G   E   R   A   T   L   S   C   R   A   S|tcg|cta|agc|ccg|ggt|gaa|cgt|gct|acc|tta|agt|tgc|cgt|gct|tcc|   EspI.....                       AflII...            XmaI.... (CDR1installed as AflII-(SexAI or KasI) cassette.) For the most preferred 11length codon 51 (XXX) is omitted; for the preferred 12 length this codonis <2> -------- CDR1 --------------------->|--- FR2 -------------->     <1>     <2> <2> xxx <3> 46  47  48  49  50  51  52  53  54  55  56  57  58  59  60  Q   -   V   -   -   -   -  L   A   W   Y   Q   Q   K   P |cag| -|gtt| - | - | - | - |ctt|gct|tgg|tat|caa|cag|aaa|cct|                                                      SexAI... CDR2installed as (SexAI or KasI) to (BamHI or RsrII) cassette.) ---- FR2------------------------->|------- CDR2 ---------->                                   <1>          <2>     <4> 61  62  63  64  65  66  67  68  69  70  71  72  73  74  75  G   Q   A   P   R   L   L   I   Y   -   A   S   -   R   -|ggt|cag|gcg|ccg|cgt|tta|ctt|att|tat| - |gct|tct| - |cgc| - |SexAI....  KasI.... CDR2-->|--- FR3------------------------------------------->  <1> 76  77  78  79  80  81  82  83  84  85  86  87  88  89  90  -   G   I   P   D   R   F   S   G   S   G   S   G   T   D | -|ggg|atc|ccg|gac|cgt|ttc|tct|ggc|tct|ggt|tca|ggt|act|gac|       BamHI...             RsrII..... ------ FR3------------------------------------------------> 91  92  93  94  95  96  97  98  99 100 101 102 103 104 105  F   T   L   T   I   S   R   L   E   P   E   D   F   A   V|ttt|acc|ctt|act|att|tct|aga|ttg|gaa|cct|gaa|gac|ttc|gct|gtt|                     XbaI... For CDR3 (Length 8): QQ33111P 1 and 3 asdefined for Length 9 For CDR3 (Length 10): QQ3211PP1T 1 and 3 as definedfor Length 9 2 S (0.2) and 0.044 each of ADEFGHIKLMNPQRTVWY CDR3installed as XbaI to (StyI or BsiWI) cassette.----------->|----CDR3-------------------------->|-----FR4--->                     <3> <1> <1> <1>     <1> 106 107 108 109 110 111 112113 114 115 116 117 118 119 120  Y   Y   C   Q   Q   -   -   -   -   P   -   T   F   G   Q|tat|tat|tgc|caa|cag| - | - | - | - |cct| - |act|ttc|ggt|caa|           BstXI.......... -----FR4------------------->|      <--------Ckappa ----------- 121 122 123 124 125 126 127        128 129 130 131132 133 134  G   T   K   V   E   I   K         R   T   V   A   A   P   S|gt|acc|aag|gtt|gaa|atc|aag|      |cgt|acg|gtt|gcc|gct|cct|agt|     StyI....                      BsiWI.. 135 136 137 138 139 140 141142 143 144 145 146 147 148 149  V   F   I   F   P   P   S   D   E   Q   L   K   S   G   T|gtg|ttt|atc|ttt|cct|cct|tct|gac|gaa|caa|ttg|aag|tca|ggt|act|                                     MfeI... 150 151 152 153 154 155 156157 158 159 160 161 162 163 164  A   S   V   V   C   L   L   N   N   F   Y   P   R   E   A|gct|tct|gtc|gta|tgt|ttg|ctc|aac|aat|ttc|tac|cct|cgt|gaa|gct|                                              BssSI... 165 166 167 168169 170 171 172 173 174 175 176 177 178 179  K   V   Q   W   K   V   D   N   A   L   Q   S   G   N   S|aaa|gtt|cag|tgg|aaa|gtc|gat|aac|gcg|ttg|cag|tcg|ggt|aac|agt|                              MluI.... 180 181 182 183 184 185 186 187188 189 190 191 192 193 194  Q   E   S   V   T   E   Q   D   S   K   D   S   T   Y   S|caa|gaa|tcc|gtc|act|gaa|cag|gat|agt|aag|gac|tct|acc|tac|tct| 195 196197 198 199 200 201 202 203 204 205 206 207 208 209  L   S   S   T   L   T   L   S   K   A   D   Y   E   K   H|ttg|tcc|tct|act|ctt|act|tta|tca|aag|gct|gat|tat|gag|aag|cat| 210 211212 213 214 215 216 217 218 219 220 221 222 223 224  K   V   Y   A   C   E   V   T   H   Q   G   L   S   S   P|aag|gtc|tat|GCt|TGC|gaa|gtt|acc|cac|cag|ggt|ctg|agc|tcc|cct|                                               SacI.... 225 226 227 228229 230 231 232 233 234   V   T   K   S   F   N   R   G   E   C   .   .|gtt|acc|aaa|agt|ttc|aac|cgt|ggt|gaa|tgc|taa|tag ggcgcgcc                       DsaI....                  AscI....                                                  BssHII acgcatctctaagcggccgc aacaggaggag              NotI....For CDR1:<1> ADEFGHIKLMNPQRSTVWY 1:1<2> S (0.2) ADEFGHIKLMNPQRTVWY (0.044 each)<3> Y (0.2) ADEFGHIKLMNPQRSTVW (0.044 each)For CDR2:<1> ADEFGHIKLMNPQRSTVWY 1:1<2> S (0.2) ADEFGHIKLMNPQRTVWY (0.044 each)<4> A (0.2) DEFGHIKLMNPQRSTVWY (0.044 each)For CDR3 (Length 9):<1> ADEFGHIKLMNPQRSTVWY 1:1<3> Y (0.2) ADEFGHIKLMNPQRTVW (0.044 each)

TABLE 8 2a2 JH2 Human lambda-chain gene gaggaccatt gggcccc ttactccgtgacScab...... Eco0109I            ApaI..        -----------FR1-------------------------------------------->         1   2   3   4   5   6   7   8   9  10  11  12  13  14  15 S   A   Q   S   A   L   T   Q   P   A   S   V   S   G   S   P   Gagt|gca|caa|tcc|gct|ctc|act|cag|cct|gct|agc|gtt|tcc|ggg|tca|cct|ggt| ApaLI...                           NheI...          BstEII...                                                          SexAI.....                              T   G  <1>  S   S  <2>  V   G------FR1------------------> |-----CDR1-------------------- 16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  Q   S   I   T   I   S   C   T   G   -   S   S   -   V   G|caa|agt|atc|act|att|tct|tgt|aca|ggt| - |tct|tct| - gtt|ggc|                         BsrGI..  <1> <3> <2> <3>  V   S = vg Scheme #1,length = 14 -----CDR1------------->|--------FR2------------------------- 31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  -   -   -   -   V   S   W   Y   Q   Q   H   P   G   K   A | - | - | -| - |gtt|tct|tgg|tat|caa|caa|cac|ccg|ggc|aag|gcg|                                           XmaI....    KasI                                           AvaI....                         <4> <4> <4> <2>  R   P   S--FR2------------------> |------CDR2--------------->|----FR3- 46  47  48  49  50  51  52  53  54  55  56  57  58  59  60  P   K   L   M   I   Y   -   -   -   -   R   P   S   G   V|ccg|aag|ttg|atg|atc|tac| - | - | - | - |cgt|cct|tct|ggt|gtt| KasI....FR3  61  62  63  64  65  66  67  68  69  70  71  72  73  74  75  S   N   R   F   S   G   S   K   S   G   N   T   A   S   L|agc|aat|cgt|ttc|tcc|gga|tct|aaa|tcc|ggt|aat|acc|gca|agc|tta|                 BspEI..                           HindIII.                      BsaBI........(blunt)-------FR3------------------------------------------------->1 76  77  78  79  80  81  82  83  84  85  86  87  88  89  90  T   I   S   G   L   Q   A   E   D   E   A   D   Y   Y   C|act|atc|tct|ggt|ctg|cag|gct|gaa|gac|gag|gct|gac|tac|tat|tgt|                 PstI... <4> <5> <4> <2> <4> S <4> <4> <4> <4> V-----CDR3-------------------------------->|---FR4---------- 91  92  93  94  95  96  97  98  99 100 101 102 103 104 105  -   -   -   -   -   S   -   -   -   -   V   F   G   G   G | - | - | -| - | - |tct| - | - | - | - |gtc|ttc|ggc|ggt|ggt|                                                         KpnI...------FR4--------------> 106 107 108 109 110 111 112 113 114 115 116 117118 119 120   T   K   L   T   V   L   G   Q   P   K   A   A   P   S   V|acc|aaa|ctt|act|gtc|ctc|ggt|caa|cct|aag|gct|gct|cct|tcc|gtt|KpnI...                   HincII..                                Bsu36I... 121 122 123 124 125 126 127128 129 130 131 132 133 134 135  T   L   F   P   P   S   S   E   E   L   Q   A   N   K   A|act|ctc|ttc|cct|cct|agt|tct|gaa|gag|ctt|caa|gct|aac|aag|gct|                             SapI...... 136 137 138 139 140 141 142 143144 145 146 147 148 149 150  T   L   V   C   L   I   S   D   F   Y   P   G   A   V   T|act|ctt|gtt|tgc|ttg|atc|agt|gac|ttt|tat|cct|ggt|gct|gtt|act|                  BclI.... 151 152 153 154 155 156 157 158 159 160 161162 163 164 165  V   A   W   K   A   D   S   S   P   V   K   A   G   V   E|gtc|gct|tgg|aaa|gcc|gat|tct|tct|cct|gtt|aaa|gct|ggt|gtt|gag|                                                         BsmBI... 166167 168 169 170 171 172 173 174 175 176 177 178 179 180  T   T   T   P   S   K   Q   S   N   N   K   Y   A   A   S|acg|acc|act|cct|tct|aaa|caa|tct|aac|aat|aag|tac|gct|gcg|agc|BsmBI....                                              SacI.... 181 182183 184 185 186 187 188 189 190 191 192 193 194 195  S   Y   L   S   L   T   P   E   Q   W   K   S   H   K   S|tct|tat|ctt|tct|ctc|acc|cct|gaa|caa|tgg|aag|tct|cat|aaa|tcc| SacI...196 197 198 199 200 201 202 203 204 205 206 207 208 209 210  Y   S   C   Q   V   T   H   E   G   S   T   V   E   K   T|tat|tcc|tgt|caa|gtt|act|cat|gaa|ggt|tct|acc|gtt|gaa|aag|act|                      BspHI... 211 212 213 214 215 216 217 218 219  V   A   P   T   E   C   S   .   .|gtt|gcc|cct|act|gag|tgt|tct|tag|tga|ggcgcgcc                                    AscI....                                     BssHII aacgatgttc aag gcggccgcaacaggaggag                NotI.... Scab.......For CDR1 (length 14):<1> = 0.27 T, 0.27 G, 0.027 each of ADEFHIKLMNPQRSVWY, no C<2> = 0.27 D, 0.27 N, 0.027 each of AEFGHIKLMPQRSTVWY, no C<3> = 0.36 Y, 0.0355 each of ADEFGHIKLMNPQRSTVW, no CA second Vg scheme for CDR1 gives segments of length 11:T₂₂G<2><4>L<4><4><4><3><4><4> where<4> 32 e|uimolar mixture of each of ADEFGHIKLMNPQRSTVWY, no C<3> 32 as defined above for the alternative CDR1For CDR2:<2> and <4> are the same variegation as for CDR1CDR3 (Length 11):<2> and <4>are the same variegation as for CDR1<5> = 0.36 S, 0.0355 each of ADEFGHIKLMNPQRTVWY no CCDR3 (Length 10): <5> SY <1> <5> S <5> <1> <4> V<1> is an equimolar mixture of ADEFGHIKLMNPQRSTVWY, no C<4> and <5> are as defined for Length 11

TABLE 9 Oligonucleotides For Kappa and Lambda Light Chain Variegation(Ctop25): 5′-gctctggtcaac|tta|agg|gct|gag|g-3′ (CtprmA):5′-gctctggtcaac|tta|agg|gct|gag|gac|acc|gct|gtc|tac|tac|tgc|gcc-3′              AflII... (CBprmB) [RC]:5′-|tac|ttc|gat|tac|ttg|ggc|caa|ggt|acc|ctg|gtc|acc|tcgctccacc-3′                                          BstEII... (CBot25) [RC]:5′-|ggt|acc|ctg|gtc|acc|tcgctccacc-3′ Kappa chains: CDR1 (“1”), CDR2(“2”), CDR3 (“3”) CDR1 (Ka1Top610):5′-ggtctcagttg|cta|agc|ccg|ggt|gaa|cgt|gct|acc|tta|agt|tgc|cgt|gct|tcc|cag-3′(Ka1STp615): 5′-ggtctcagttg|cta|agc|ccg|ggt|g-3′ (Ka1Bot620) [RC]:5′-ctt|gct|tgg|tat|caa|cag|aaa|cct|ggt|cag|gcg|ccaagtcgtgtc-3′(Ka1SB625) [RC]: 5′-cct|ggt|cag|gcg|ccaagtcgtgtc-3′ (Ka1vg600):5′-gct|acc|tta|agt|tgc|cgt|gct|tcc|cag-|<1>|gtt|<2>|<2>|<3>|ctt|gct|tgg|tat|caa|cag|aaa|cc-3′ (Ka1vg600-12):5′-gct|acc|tta|agt|tgc|cgt|gct|tcc|cag-|<1>|gtt<2>|<2>|<2>|<3>|ctt|gct|tgg|tat|caa|cag|aaa|cc-3′ CDR2(Ka2Tshort657): 5′-cacgagtccta|cct|ggt|cag|gc-3′ (Ka2Tlong655):5′-cacgagtccta|cct|ggt|cag|gdg|ccg|cgt|tta|ctt|att|tat-3′(Ka2Bshort660): [RC]: 5′-|gac|cgt|ttc|tct|ggt|tctcacc-3′|cgc|<4>|<1>|ggg|atc|ccg|gac|cgt|ttc|tct|ggt|tctcacc-3′ CDR3(Ka3Tlon672):5′-gacgagtccttct|aga|ttg|gaa|cct|gaa|gac|ttc|gct|gtt|tat|tat|tgc|caa|c-3′(Ka3BotL682) [RC]:5′-act|ttc|ggt|caa|ggt|acc|aag|gtt|gaa|atc|aag|cgt|acg|tcacaggtgag-3′(Ka3Bsho694) [RC]: 5′-gaa|atc|aag|cgt|acg|tcacaggtgag-3′ (Ka3vg670):5′-gac|ttc|gct|gtt|-|tat|tat|tgc|caa|cag|<3>|<1>|<1>a<1>|cct|<1>|act|ttc|ggt|caa|-|ggt|acc|aag|gtt|g-3′ (Ka3vg670-8): 5′-gac|ttc|gct|gtt|-|tat|tat|tgc|caa|cag|<3>|<3>|<1>|<1>|<1>|cct|ttc|ggt|caa|-|ggt|acc|aag|gtt|g-3′ (Ka3vg670-10): 5′-gac|ttc|gct|gtt|tat|-|tat|tgc|caa|cag|<3>|<2>|<1>|<1>|cct|cct|<1>|act|ttc|ggt|caa|-|ggt|acc|aag|gtt|g-3′ Lambda Chains: CDR1 (“1”), CDR2 (“2”), CDR3 (“3”)CDR1 (Lm1TPri75): 5′-gacgagtcctgg|tca|cct|ggt|-3′ (Lm1tlo715):5′-gacgagtcctgg|tca|cct|ggt|caa|agt|atc|act|att|tct|tgt|aca|ggt-3′(Lm1blo724) [rc]:5′-gtt|tct|tgg|tat|caa|caa|cac|ccg|ggc|aag|gcg|agatcttcacaggtgag-3′(Lm1bsh737) [rc]: 5′-gc|aag|gcg|agatcttcacaggtgag-3′ (Lm1vh710b):5′-gt|atc|act|att|tct|tgt|aca|ggt|<2>|<4>|ctc|<4>|<4>|<4>|-|<3>|<4>|<4>|tgg|tat|caa|caa|cac|cc-3-′ (Lm1vh710):5′-gt|atc|act|att|tct|tgt|aca|ggt|<1>|tct|tct|<2>|gtt|ggc|-|<1>|<3>|<2>|<3>|gtt|tct|tgg|tat|caa|caa|cac|cc-3′ CDR2 (Lm2TSh757):5′-gagcagaggac|ccg|ggc|aag|gc-3′ (Lm2TLo753):5′-gagcagaggac|ccg|ggc|aag|gcg|ccg|aag|ttg|atg|atc|tac|-3′ (Lm2BLo762)[RC]: 5′-cgt|cct|tct|ggt|gtc|agc|aat|cgt|ttc|tcc|gga|tcacaggtgag-3′(Lm2BSh765) [RC]: 5′-cgt|ttc|tcc|gga|tcacaggtgag-3′ (Lm2vg750):5′-g|ccg|aag|ttg|atg|atc|tac|-<4>|<4>|<4>|<2>|cgt|cct|tct|ggt|gtc|agc|aat|c-3′ CDR3 (Lm3TSh822):5′-ctg|cag|gct|gaa|gac|gag|gct|gac-3′ (Lm3TLo819):5′-ctg|cag|gct|gaa|gac|gag|gct|gac|tac|tat|tgt|-3′ (Lm3BLo825) [RC]:5′-gtc|ttc|ggc|ggt|ggt|acc|aaa|ctt|act|gtc|ctc|ggt|caa|cct|aag|g-acacaggtgag-3′ (Lm3BSh832) [RC]: 5′-c|ggt|caa|cct|aag|gacacaggtgag-3′(Lm3vg817): 5′-gac|gag|gct|gac|tac|tat|tgt|-|<4>|<5>|<4>|<2>|<4>|tct|<4>|<4>|<4>|<4>|-Gtc|ttc|ggc|ggt|ggt|acc|aaa|ctt|ac-3′ (Lm3vg817-10): 5′-gac|gag|gct|gac|tac|tat|tgt|-|<5>|agc|tat|<1>|<5>|tct<5>|<1>|<4>|gtc|ttc|ggc|ggt|ggt|-|acc|aaa|ctt|ac-3′

TABLE 10 A27:JH1 Kappa light chain gene with stuffers in place of CDRsEach stuffer contains at least one stop codon and a restriction sitethat will be uni|ue within the diversity vector. gaggacc attgggccccctccgagact ctcgagcgca   Scab.....Eco0109I            ApaI.                             XhoI.. acgcaattaa tgtgagttag ctcactcattaggcacccca ggctttacac tttatgcttc      ..-35..          Plac                    ..-10. cggctcgtatgttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatgac catgattacgccaagctt tggagccttt tttttggaga ttttcaac           PflMI.......            Hind3. M13 III signal se|uence (AAse|)--------------------------> 1   2   3   4   5   6   7   8   9  10  11  12  13  14  15 M   K   K   L   L   F   A   I   P   L   V   V   P   F   Y gtg aag aagctc cta ttt gct atc ccg ctt gtc gtt ccg ttt tac--Signal-->  FR1-------------------------------------------> 16  17  18  19  20  21  22  23  24  25  26  27  28  29  30  S   H   S   A   Q   S   V   L   T   Q   S   P   G   T   L|agc|cat|agt|gca|caa|tcc|gtc|ctt|act|caa|tct|cct|ggc|act|ctt|          ApaLI... ----- FR1--------------------------------->|-------Stuffer-> 31  32  33  34  35  36  37  38  39  40  41  42  43  S   L   S   P   G   E   R   A   T   L   S   |   ||tcg|cta|agc|ccg|ggt|gaa|cgt|gct|acc|tta|agt|tag|taa|gct|ccc|   EspI.....                       AflII...            XmaI.... -Stuffer for CDR1-->FR2 --------- FR2 ------>|-----------Stuffer for CDR2                        59  60  61  62  63  64  65  66                         K   P   G   Q   A   P   R|agg|cct|actt|tga|tct|g|aaa|cct|ggt|cag|gcg|ccg|cgt|taa|tga|aagcgctaatggccaacagtg StuI...                  SexAI...    KasI....              AfeI..   MscI..Stuffer-->|--- FR3 -----------------------------------------> 76  77  78  79  80  81  82  83  84  85  86  87  88  89  90  T   G   I   P   D   R   F   S   G   S   G   S   G   T   D|act|ggg|atc|ccg|gac|ccgt|ttc|tct|ggc|tct|ggt|tca|ggt|act|gac|     BamHI...             RsrII..... ------ FR3----->----------------STUFFER for CDR3-----------------> 91  92  93  94  95  96  97   F   T   L   T   I   S   R   |   ||ttt|acc|ctt|act|att|tct|aga|taa|tga| gttaac tag acc tacgta acc tag                     XbaI...          HpaI..         SnaBI.-----------------CDR3 stuffer------------------>|-----FR4--->                                                118 119 120                                                  F   G   Q                                                |ttc|ggt|caa|-----FR4------------------->|     <------ Ckappa ------------- 121 122123 124 125 126 127       128 129 130 131 132 133 134  G   T   K   V   E   I   K        R   T   V   A   A   P   S|ggt|acc|aag|gtt|gaa|atc|aag|    |cgt|acg|gtt|gcc|gct|cct|agt|      StyI....                    BsiWI.. 135 136 137 138 139 140 141142 143 144 145 146 147 148 149  V   F   I   F   P   P   S   D   E   Q   L   K   S   G   T|gtg|ttt|atc|ttt|cct|cct|tct|gac|gaa|caa|ttg|aag|tca|ggt|act|                                     MfeI... acgcatctctaa gcggccgcaacaggaggag             NotI....              EagI..

TABLE 11 2a2:JH2 Human lambda-chain gene with stuffers in place of CDRsgaggaccatt gggcccc ttactccgtgac Scab...... Eco0109I            ApaI..         ----------FR1-------------------------------------------->         1   2   3   4   5   6   7   8   9  10  11  12  13  14  15 S   A   Q   S   A   L   T   Q   P   A   S   V   S   G   S   P   Gagt|gca|caa|tcc|gct|ctc|act|cag|cct|gct|agc|gtt|tcc|ggg|tca|cct|ggt| ApaLI...                           NheI...          BstEII...                                                          SexAI....------FR1------------------> |-----stuffer for CDR1--------- 16  17  18  19  20  21  22  23   Q   S   I   T   I   S   C   T|caa|agt|atc|act|att|tct|tgt|aca|tct tag tga ctc                         BsrGI..-----Stuffer--------------------------->-------FR2----------> 31  32  33  34  35  36  37  38  39  40  41  42  43  44  45  R   S   |   |   P   |                   H   P   G   K   A aga tct taatga ccg tag                 cac|ccg|ggc|aag|gcg|BglII                                     XmaI....     KasI.....                                          AvaI....      --|--------------Stuffer for CDR2 ----------------------------->       P      |ccg|taa|tga|atc tcg tacg                        ct|ggt|gtt| KasI....               BsiWI...-------FR3---------------------------------------------------- 61  62  63  64  65  66  67  68  69  70  71  72  73  74  75  S   N   R   F   S   G   S   K   S   G   N   T   A   S   L|agc|aat|cgt|ttc|tcc|gga|tct|aaa|tcc|ggt|aat|acc|gca|agc|tta|                 BspEI..                           HindIII.                      BsaBI........(blunt)-------FR3------------->|--Stuffer for CDR3---------------->| 76  77  78  79  80  81  82  83  84  85  86  87  88  89  90  T   I   S   G   L   Q |act|atc|tct|ggt|ctg|caglgtt ctg tag ttc caattgctt tag tga ccc                  PstI...                 MfeI..-----Stuffer------------------------------->|---FR4---------                                                103 104 105                                                 G   G   G                                               |ggc|ggt|ggt|                                                         KpnI...---------FR4-------------->    106 107 108 109 110 111 112 113 114 115116 117 118 119 120     T   K   L   T   V   L   G   Q   P   K   A   A   P   S   V   |acc|aaa|ctt|act|gtc|ctc|ggt|caa|cct|aag|gct|gct|cct|tcc|gtt|KpnI...                      HincII..                                    Bsu36I...  121 122 123 124 125 126127 128 129 130 131 132 133 134 135  T   L   F   P   P   S   S   E   E   L   Q   A   N   K   A|act|ctc|ttc|cct|cct|agt|tct|gaa|gag|ctt|caa|gct|aac|aag|gct|                             SapI.....  136 137 138 139 140 141 142 143144 145 146 147 148 149 150  T   L   V   C   L   I   S   D   F   Y   P   G   A   V   T|act|ctt|gtt|tgc|ttg|atc|agt|gac|ttt|tat|cct|ggt|gct|gtt|act|                  BclI....

1. A focused library of vectors or genetic packages that display,display and express, or comprise a member of a diverse family of humanantibody related peptides, polypeptides and proteins and collectivelydisplay, display and express, or comprise at least a portion of thediversity of the antibody family, the vectors or genetic packages beingcharacterized by variegated DNA sequences that encode a heavy chain CDR1selected from the group consisting of: (1) <1>₁Y₂<1>₃M₄<1>₅, wherein <1>is an equimolar mixture of each of amino acid residues A, D, E, F, G, H,I, K, L, M, N, P, Q, R, S, T, V, W, and Y; (2)(S/T)₁(S/G/X)₂(S/G/X)₃Y₄Y₅W₆(S/G/X)₇. wherein (S/T) is a 1:1 mixture ofS and T residues, (S/G/X) is a mixture of 0.2025 S, 0.2025, G and 0.035of each of amino acid residues A, D, E, F, H, I, K, L, M, N, P, Q, R, T,V, W, and Y; (3) V₁S₂G₃G₄S₅I₆S₇<<1>₈<1>₉<1>₁₀Y₁₁Y₁₂W₁₃<1>₁₄, wherein <1>is an equimolar mixture of each of amino acid residues A, D, E, F, G,H,. I, K, L, M, N, P, Q, R, S, T, V, W, and Y; and (4) mixtures ofvectors or genetic packages characterized by any of the above DNAsequences.
 2. The focused library according to claim 1, wherein HC CDR1s(1), (2) and (3) are present in the library in the ratio 0.80:0.17:0.02.3. A focused library of vectors or genetic packages that display,display and express, or comprise a member of a diverse family of humanantibody related peptides, polypeptides and proteins and collectivelydisplay, display and express, or comprise at least a portion of thediversity of the antibody facility, the vectors or genetic packagesbeing characterized by variegated DNA sequences that encode a heavychain CDR2 selected from the group consisting of: (1)<2>I<2><3>SGG<1>T<1>YADSVKG, wherein <1> is an equimolar mixture of eachof amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,V, W, and Y; <2> is an equimolar mixture of each of amino acid residuesY, R, W, V, G, and S; and <3> is an equimolar mixture of each of aminoacid residues P, S, and G or an equimolar mixture of P and S; (2)<1>I<4><1><1><G><5><1><1><1>YADSVKG, wherein <1> is an equimolar mixtureof each of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W, and Y; <4> is an equimolar mixture of residues D, I, N, S,W, Y; and <5> is an equimolar mixture of residues S, G, D and N; (3)<1>I<4><1><1>G<5><1><1>YNPSLKG, wherein <1> is an equimolar mixture ofeach of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W and Y; and <4> and <5> are as defined above; (4)<1>I<8>S<1><1><1>GGYY<1>YAASVKG, wherein <1> is an equimolar mixture ofeach amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,V, W and Y; <8> is 0.27 R and 0.027 of each of ADEFGHIKLMNPQSTVWY; and(5) mixtures of vectors or genetic packages characterized by any of theabove DNA sequences.
 4. The focused library according to claim 3 whereina mixture of HC CDR2s (1)/(2) (equimolar), (3) and (4) are present inthe library in a ratio of 0.54:0.43:0.03.
 5. A focused library ofvectors or genetic packages that display, display and express, orcomprise a member of a diverse family of human antibody relatedpeptides, polypeptides and proteins and collectively display, displayand express, or comprise at least a portion of the diversity of theantibody family, the vectors or genetic packages being characterized byvariegated DNA sequences that encode a heavy chain CDR3 selected fromthe group consisting of: (1) YYCA21111YFDYWG, wherein 1 is an equimolarmixture of each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P,Q, R, S, T, V, W and Y; and 2 is an equimolar mixture of K and R; (2)YYCA2111111YFDYWG, wherein 1 is an equimolar mixture of each amino acidresidues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and2 is an equimolar mixture of K and R; (3) YYCA211111111YFDAYTG, wherein1 is an equimolar mixture of each amino acid residues A, D, E, F, G, H,I, K, L, M, N, P, Q, R, S, T, V, W and Y; and 2 is an equimolar mixtureof K and R; (4) YYCAR111S2S3111YFDYWG, wherein 1 is an equimolar mixtureof each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W and Y; and 2 is an equimolar mixture of S and G; and 3 is anequimolar mixture of Y and W; (5) YYCA2111CSG11CY1YFDYWG, wherein 1 isan equimolar mixture of each amino acid residues A, D, E, F, G, H, I, K,L, M, N, P, Q, R, S, T, V, W and Y; and 2 is an equimolar mixture of Kand R; (6) YYCA211S1TIFG11111YFDYWG, wherein 1 is an equimolar mixtureof each amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,T, V, W and Y; and 2 is an equimolar mixture of K and R; (7)YYCAR111YY2S3344111YFDYWG, wherein 1 is an equimolar mixture of eachamino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Wand Y; 2 is an equimolar mixture of D and S; and 3 is an equimolarmixture of S and G; (8) YYCAR1111YC2231CY111YFDYWG, wherein 1 is anequimolar mixture of each amino acid residues A, D, E, F, G, H, I, K, L,M, N, P,. Q, R, S, T, V, W and Y; 2 is an equimolar mixture of S and G;and 3 is an equimolar mixture of T, D and G; and (9) mixtures of vectorsor genetic packages characterized by any of the above DNA sequences. 6.The focused library according to claim 5, wherein 1 in one or all of HCCDR3s (1) through (8) is 0.095 of each of G and Y and 0.048 of each ofA, D, E, F, H, I, K, L, M, N, P, Q, R, S, T, V, and W.
 7. The focusedlibrary according to claim 5 or 6, wherein HC CDR3s (1) through (8) arepresent in the library in the following proportions: (1) 0.10 (2) 0.14(3) 0.25 (4) 0.13 (5) 0.13 (6) 0.11 (7) 0.04 and (8) 0.10
 8. The focusedlibrary according to claim 5 or 6, wherein the HC CDR3s (1) through (8)are present in the library in the following proportions: (1) 0.02 (2)0.14 (3) 0.25 (4) 0.14 (5) 0.14 (6) 0.12 (7) 0.08 and (8) 0.11
 9. Afocused library of vectors or genetic packages that display, display andexpress, or comprise a member of a diverse family of human antibodyrelated peptides, polypeptides and proteins and collectively display,display and express, or comprise at least a portion of the diversity ofthe antibody family, the vectors or genetic packages being characterizedby variegated DNA sequences that encodes a kappa light chain CDR1selected from the group consisting of: (1) RASQ<1>V<2><2><3>LA (2)RASQ<1>V<2><2><2><3>LA; wherein <1> is an equimolar mixture of aminoacid residues ADEFGHIKLMNPQRSTVWY; <2> is 0.2 S and 0.044 of each ofADEFGHIKLMNPQRTVWY; and <3> is 0.2Y and 0.044 each of ADEFGHIKLMNPQRTVWand Y; and (3) mixtures of vectors or genetic packages characterized byany of the above DNA sequences.
 10. The focused library of claim 9,wherein CDR1s (1) and (2) are present in the library in a ratio of0.68:0.32.
 11. A focused library of vectors or genetic packages thatdisplay, display and express, or comprise a member of a diverse familyof human antibody related peptides, polypeptides and proteins andcollectively display, display and express, or comprise at least aportion of the diversity of the antibody family, the vectors or geneticpackages being characterized by variegated DNA sequences that encode akappa light chain CDR2 having the sequence: <1>AS<2>R<4><1>, wherein <1>is an equimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVWY; <2>is 0.2 S and 0.044 of each of ADEFGHIKLMNPQRTVWY; and <4> is 0.2 A and)0.044 each of DEFGHIKLMNPQRSTVWY.
 12. A focused library of vectors orgenetic packages that display, display and express, or comprise a memberof a diverse family of human antibody related peptides, polypeptides andproteins and collectively display, display and express, or comprise atleast a portion of the diversity of the antibody family, the vectors orgenetic packages being characterized by variegated DNA sequences thatencode a kappa light chain CDR3 selected from the groups consisting of:(1) QQ<3><1><1><1>P<1>T, wherein <1> is an equimolar mixture of aminoacid residues ADEFGHIKLMNPQRSTVWY; <3> is 0.2 Y and 0.044 each ofADEFGHIKLMNPQRTVW; (2) QQ33111P, wherein 1 and 3 are as defined in (1)above; (3) QQ3211PP1T, wherein 1 and 3 are as defined in (1) above and 2is 0.2 S and 0.044 each of ADEFGHIKLMNPQRTVWY; and (4) mixtures ofvectors or genetic packages characterized by any of the above DNAsequences.
 13. The focused library according to claim 12, wherein CDR3s(1), (2) and (3) are present in the library in a ratio of 0.65:0.1:0.25.14. A focused library of vectors or genetic packages that display,display and express, or comprise a member of a diverse family of humanantibody related peptides, polypeptides and proteins and collectivelydisplay, display and express, or comprise at least a portion of thediversity of the antibody family, the vectors or genetic packages beingcharacterized by variegated DNA sequences that encode a lambda lightchain CDR1 selected from the group consisting of: (1)TG<1>SS<2>VG<1><3><2><3>VS, wherein <1> is 0.27 T, 0.27 G and 0.027 eachof ADEFHIKLMNPQRSVWY, <2> is 0.27 D, 0.27 N and 0.027 each ofAEFGHIKLMPQRSTVWY, and <3> is 0.36 Y and 0.036 each ofADEFGHIKLMNPQRSTVW; (2) G<2><4>L<4><4><4><3><4><4>, wherein <2> is asdefined in (1) above and <4> is an equimolar mixture of amino acidresidues ADEFGHIKLMNPQRSTVWY; and (3) mixtures of vectors or geneticpackages characterized by any of the above DNA sequences.
 15. Thefocused library according to claim 14, where CDR1s (1) and (2) arepresent in the library in a ratio of 0.67:0.33.
 16. A focused library ofvectors or genetic packages that display, display and express, orcomprise a member of a diverse family of human antibody relatedpeptides, polypeptides and proteins and collectively display, displayand express, or comprise at least a portion of the diversity of theantibody family, the vectors or genetic packages being characterized byvariegated DNA sequences that encode a lambda light chain CDR2 has thesequence: <4><4><4><2>RPS, wherein <2> is 0.27 D, 0.27 N, and 0.027 eachof AEFGHIKLMPQRSTVWY and <4> is an equimolar mixture of amino acidresidues ADEFGHIKLMNPQRSTVW.
 17. A focused library of vectors or geneticpackages that display, display and express, or comprise a member of adiverse family of human antibody related peptides, polypeptides andproteins and collectively display, display and express, or comprise atleast a portion of the diversity of the antibody family, the vectors orgenetic packages being characterized by variegated DNA sequences thatencode a lambda light chain CDR3 selected from the group consisting of:(1) <4><5><4><2><4>S<4><4><4><4>V, wherein <2> is 0.27 D, 0.27 N, and0.027 each of AEFGHIKLMPQRSTVWY; <4> is an equimolar mixture of aminoacid residues ADEFGHIKLMNPQRSTVW; and <5> is 0.36 S and 0.0355 each ofADEFGHIKLMNPQRTVWY; (2) <5>SY<1><5>S<5><1><4>V, wherein <1> is anequimolar mixture of ADEFGHIKLMNPQRSTVWY; and <4> and <5> are as definedin (1) above; and (3) mixtures of vectors or genetic packagescharacterized by any of the above DNA sequences.
 18. The focused libraryaccording to claim 17, wherein CDR3s (1) and (2) are present in thelibrary in an equimolar mixture.
 19. The focused library according toclaim 1 or 2 further comprising variegated DNA sequences that encode aheavy chain CDR selected from the group consisting of: (1) one or moreof the heavy chain CDR2s defined in claim 3 or 4; (2) one or more of theheavy chain CDR3s defined in claims 5, 6, 7, or 8; and (3) mixtures ofvectors or genetic packages characterized by (1) and (2).
 20. Thefocused library according to claim 3 further comprising variegated DNAsequences that encodes one or more heavy chain CDR3s selected from thegroup defined in claims 5, 6, 7 or
 8. 21. The focused library accordingto claim 19 or 20, further comprising variegated DNA sequences thatencodes a light chain CDR selected from the group consisting of (1) oneor more the kappa light chain CDR1s defined in claim 9 or 10; (2) thekappa light chain CDR2 defined in claim 11; (3) one or more of the kappalight chain CDR3s defined in claim 12 or 13; (4) one or more of thekappa light chain CDR1s defined in claim 14 or 15; (5) the lambda lightchain CDR2 defined in claim 16; (6) one or more of the lambda lightchain CDR3s defined in claim 17 or 18; and (7) mixtures of vectors andgenetic packages characterized by one or more of (1) through (6).
 22. Apopulation of variegated DNA sequences that encode a heavy chain CDR1selected from the group consisting of: (1) <1>₁Y₂<1>₃M₄<1>₅, wherein <1>is an equimolar mixture of each of amino acid residues A, D, E, F, G, H,I, K, L, M, N, P, Q, R, S, T, V, W, and Y; (2) (S/T)₁(S/G/X)₂(S/G/X)₃Y₄Y₅W₆(S/G/X)₇ wherein (S/T) is a 1:1 mixture of S and Tresidues, (S/G/X) is a mixture of 0.2025 S, 0.2025 G and 0.035 of eachof amino acid residues A, D, E, F, H, I, K, L, M, N, P, Q, R, T, V, W,and Y; (3) V₁S₂G₃G₄S₅I₆S₇<1>₈<1>₉<1>₁₀Y₁₁Y₁₂W₁₃<1>₁₄, wherein <1> is anequimolar mixture of each of amino acid residues A, D, E, F, G, H, I, K,L, M,. N, P, Q, R, S, T, V, W, and Y; and (4) mixtures of variegated DNAsequences characterized by any of the above DNA sequences.
 23. Thepopulation of variegated DNA sequences according to claim 22, wherein HCCDR1s (1), (2) and (3) are present in the population in the ratio0.80:0.17:0.02.
 24. A population of variegated DNA sequences that encodea heavy chain CDR2 selected from the group consisting of: (1)<2>I<2><3>SGG<1>T<1>YADSVKG, wherein <1> is an equimolar mixture of eachof amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,V, W, and Y; <2> is an equimolar mixture of each of amino acid residuesY, R, W, V, G, and S; and <3> is an equimolar mixture of each of aminoacid residues P, S, and G or an equimolar mixture of P and S; (2)<1>I<4><1><1><G><5><1><1><1>YADSVKG, wherein <1> is an equimolar mixtureof each of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R,S, T, V, W, and Y; <4> is an equimolar mixture of residues D, I, N, S,W, Y; and <5> is an equimolar mixture of residues S, G, D and N; (3)<1>I<4><1><1>G<5><1><1>YNPSLKG, wherein <1> is an equimolar mixture ofeach of amino acid residues A, D, E, F, G, H, I, K, L, M, N, P,. Q, R,S, T, V, W and Y; and <4>and <5> are as defined above; (4)<1>I<8>S<1><1><1>GGYY<1>YAASVKG, wherein <1> is an equimolar mixture ofeach amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,V, W and Y; <8> is 0.27 R and 0.027 of each of ADEFGHIKLMNPQSTVWY; and(5) mixtures of variegated DNA sequences characterized by any of theabove DNA sequences.
 25. The population of variegated DNA sequencesaccording to claim 24, wherein a mixture of HC CDR2s (1)/(2)(equimolar), (3) and (4) are present in the population in a ratio of0.54:0.43:0.03.
 26. A population of variegated DNA sequences that encodea heavy chain CDR3 selected from the group consisting of: (1)YYCA21111YFDYWG, wherein 1 is an equimolar mixture of each amino acidresidues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W and Y; and2 is an equimolar mixture of K and R; (2) YYCA2111111YFDYWG, wherein 1is an equimolar mixture of each amino acid residues A, D, E, F, G, H, I,K, L, M, N, P, Q, R, S, T, V, W and Y; and 2 is an equimolar mixture ofK and R; (3) YYCA211111111YFDAYTG, wherein 1 is an equimolar mixture ofeach amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,V, W and Y; and 2 is an equimolar mixture of K and R; (4)YYCAR111S2S3111YFDYWG, wherein 1 is an equimolar mixture of each aminoacid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W andY; and 2 is an equimolar mixture of S and G; and 3 is an equimolarmixture of Y and W; (5) YYCA2111CSG11CY1YFDYWG, wherein 1 is anequimolar mixture of each amino acid residues A, D, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W and Y; and 2 is an equimolar mixture of K andR; (6) YYCA211S1TIFG11111YFDYWG, wherein 1 is an equimolar mixture ofeach amino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T,V, W and Y; and 2 is an equimolar mixture of K and R; (7)YYCAR111YY2S3344111YFDYWG, wherein 1 is an equimolar mixture of eachamino acid residues A, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, Wand Y; 2 is an equimolar mixture of D and S; and 3 is an equimolarmixture of S and G; (8) YYCAR1111YC2231CY111YFDYWG, wherein 1 is anequimolar mixture of each amino acid residues A, D, E, F, G, H, I, K, L,M, N, P, Q, R, S, T, V, W and Y; 2 is an equimolar mixture of S and G;and 3 is an equimolar mixture of T, D and G; and (9) mixtures ofvariegated DNA sequences characterized by any of the above DNAsequences.
 27. The population of variegated DNA according to claim 26,wherein 1 in one or all of HC CDR3s (1) through (8) is 0.095 of each ofG and Y and 0.048 of each of A, D, E, F, H, I, K, L, M, N, P, Q, R, S,T, V, and W.
 28. The population of variegated DNA sequences according toclaim 26 or 27, wherein HC CDR3s (1) through (8) are present in thepopulation in the following proportions: (1) 0.10 (2) 0.14 (3) 0.25 (4)0.13 (5) 0.13 (6) 0.11 (7) 0.04 and (8) 0.10
 29. The population ofvariegated DNA sequences according to claim 26 or 27, wherein the HCCDR3s (1) through (8) are present in the population in the followingproportions: (1) 0.02 (2) 0.14 (3) 0.25 (4) 0.14 (5) 0.14 (6) 0.12 (7)0.08 and (8) 0.11
 30. A population of variegated DNA sequences thatencode a kappa light chain CDR1 selected from the group consisting of:(1) RASQ<1>V<2><2><3>LA (2) RASQ<1>V<2><2><2><3>LA; wherein <1> is anequimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVWY; <2> is 0.2S and 0.044 of each of ADEFGHIKLMNPQRTVWY; and <3> is 0.2Y and 0.044each of ADEFGHIKLMNPQRTVW and Y; and (3) mixtures of variegated DNAsequences characterized by any of the above DNA sequences.
 31. Thepopulation of variegated DNA sequences of claim 30, wherein CDR1s (1)and (2) are present in the population in a ratio of 0.68:0.32.
 32. Apopulation of variegated DNA sequences that encode a kappa light chainCDR2 having the sequence: <1>AS<2>R<4><1>, wherein <1> is an equimolarmixture of amino acid residues ADEFGHIKLMNPQRSTVWY; <2> is 0.2 S and0.044 of each of ADEFGHIKLMNPQRTVWY; and <4> is 0.2 A and) 0.044 each ofDEFGHIKLMNPQRSTVWY.
 33. A population of variegated DNA sequences thatencode a kappa light chain CDR3 selected from the groups consisting of:(1) QQ<3><1><1><1>P<1>T, wherein <1> is an equimolar mixture of aminoacid residues ADEFGHIKLMNPQRSTVWY; <3> is 0.2 Y and 0.044 each ofADEFGHIKLMNPQRTVW; (2) QQ33111P, wherein 1 and 3 are as defined in (1)above; (3) QQ3211PPlT, wherein 1 and 3 are as defined in (1) above and 2is 0.2 S and 0.044 each of ADEFGHIKLMNPQRTVWY; and (4) mixtures ofvariegated DNA sequences characterized by any of the above DNAsequences.
 34. The population of variegated DNA sequences according toclaim 33, wherein CDR3s (1) , (2) and (3) are present in the populationin a ratio of 0.65:0.1:0.25.
 35. A population of variegated DNAsequences that encode a lambda light chain CDR1 selected from the groupconsisting of: (1) TG<1>SS<2>VG<1><3><2><3>VS, wherein <1> is 0.27 T,0.2.7 G and 0.027 each of ADEFHIKLMNPQRSVWY, <2> is 0.27 D, 0.27 N and0.027 each of AEFGHIKLMPQRSTVWY, and <3> is 0.36 Y and 0.036 each ofADEFGHIKLMNPQRSTVW; (2) G<2><4>L<4><4><4><3><4><4>, wherein <2> is asdefined in (1) above and <4> is an equimolar mixture of amino acidresidues ADEFGHIKLMNPQRSTVWY; and (3) mixtures of variegated DNAsequences characterized by any of the above DNA sequences.
 36. Thepopulation of variegated DNA sequences according to claim 35, whereCDR1s (1) and (2) are present in the population in a ratio of 0.67:0.33.37. A population of variegated DNA sequences that encode a lambda lightchain CDR2 has the sequence: <4><4><4><2>RPS, wherein <2> is 0.27 D,0.27 N, and 0.027 each of AEFGHIKLMPQRSTVWY and <4> is an equimolarmixture of amino acid residues ADEFGHIKLMNPQRSTVW.
 38. A population ofvariegated DNA sequences that encode a lambda light chain CDR3 selectedfrom the group consisting of: (1) <4><5><4><2><4>S<4><4><4><4>V, wherein<2> is 0.27 D, 0.27 N, and 0.027 each of AEFGHIKLMPQRSTVWY; <4> is anequimolar mixture of amino acid residues ADEFGHIKLMNPQRSTVW; and <5> is0.36 S and 0.0355 each of ADEFGHIKLMNPQRTVWY; (2)<5>SY<1><5>S<5><1><4>V, wherein <1> is an equimolar mixture ofADEFGHIKLMNPQRSTVWY; and <4> and <5> are as defined in (1) above; and(3) mixtures of variegated. DNA sequence characterized by any of theabove DNA sequences.
 39. The population of variegated DNA sequencesaccording to claim 38, wherein CDR3s (1) and (2) are present in thepopulation in an equimolar mixture.
 40. The population of variegated DNAsequences according to claim 22 or 23 further comprising variegated DNAsequences that encode a heavy chain CDR selected from the groupconsisting of: (1) one or more of the heavy chain CDR2s defined in claim24 or 25; (2) one or more of the heavy chain CDR3s defined in claims 26,27, 28 or 29; and (3) mixtures of variegated DNA sequence characterizedby (1) and (2).
 41. The population of variegated DNA sequences accordingto claim 24 further comprising variegated DNA sequences that encodes oneor more heavy chain CDR3s selected from the group defined in claims 26,27, 28 or
 29. 42. The population of variegated DNA sequences accordingto claim 40 or 41 further comprising variegated DNA sequences thatencodes a light chain CDR selected from the group consisting of (1) oneor more the kappa light chain CDR1s defined in claim 30 or 31; (2) thekappa light chain CDR2 defined in claim 32; (3) one or more of the kappalight chain CDR3s defined in claim 33 or 34; (4) one or more of thekappa light chain CDR1s defined in claim 35 or 36; (5) the lambda lightchain CDR2 defined in claim 37; (6) one or more of the lambda lightchain CDR3s defined in claim 38 or 39; and (7) mixtures of variegatedDNA sequences characterized by one or more of (1) through (6).
 43. Apopulation of vectors comprising the variegated DNA sequences of any oneof claims 22-42.